| 著作名稱： | A Marine Terpenoid, Heteronemin, Induces Both the Apoptosis and Ferroptosis of Hepatocellular Carcinoma Cells and Involves the ROS and MAPK Pathways |
| 年度： | 2021 |
| 類別： |
期刊論文
Oxidative medicine and cellular longevity.
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| 摘要： | Hepatocellular carcinoma (HCC) is a leading cause of death, resulting in over 700 thousand deaths annually worldwide. Chemotherapy is the primary therapeutic strategy for patients with late-stage HCC. Heteronemin is a marine natural product isolated from Hippospongia sp. that has been found to protect against carcinogenesis in cholangiocarcinoma, prostate cancer, and acute myeloid leukemia. In this study, heteronemin was found to inhibit the proliferation of the HCC cell lines HA22T and HA59T and induce apoptosis via the caspase pathway. Heteronemin treatment also induced the formation of reactive oxygen species (ROS), which are associated with heteronemin-induced cell death, and to trigger ROS removal by mitochondrial SOD2 rather than cytosolic SOD1. The mitogen-activated protein kinase (MAPK) signaling pathway was associated with ROS-induced cell death, and heteronemin downregulated the expression of ERK, a MAPK that is associated with cell proliferation. Inhibitors of JNK and p38, which are MAPKs associated with apoptosis, restored heteronemin-induced cell death. In addition, heteronemin treatment reduced the expression of GPX4, a protein that inhibits ferroptosis, which is a novel form of nonapoptotic programmed cell death. Ferroptosis inhibitor treatment also restored heteronemin-induced cell death. Thus, with appropriate structural modification, heteronemin can act as a potent therapeutic against HCC. |
| 關鍵字： | Oxid Med Cell Longev . 2021 Jan 4;2021:7689045 |
| 著作名稱： | The Phenoxyphenol Compound diTFPP Mediates Exogenous C 2-Ceramide Metabolism, Inducing Cell Apoptosis Accompanied by ROS Formation and Autophagy in Hepatocellular Carcinoma Cells |
| 年度： | 2021 |
| 類別： |
期刊論文
Antioxidants
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| 摘要： | Hepatocellular carcinoma (HCC) is a severe disease that accounts for 80% of liver cancers. Chemotherapy is the primary therapeutic strategy for patients who cannot be treated with surgery or who have late-stage HCC. C2-ceramide is an effective reagent that has been found to inhibit the growth of many cancer types. The metabolism of C2-ceramide plays a vital role in the regulation of cell death/cell survival. The phenoxyphenol compound 4-{2,3,5,6-tetrafluoro-4-[2,3,5,6-tetrafluoro-4-(4-hydroxyphenoxy)phenyl]phenoxy}phenol (diTFPP) was found to have a synergistic effect with C2-ceramide, resulting in considerable cell death in the HA22T HCC cell line. diTFPP/C2-ceramide cotreatment induced a two- to threefold increase in cell death compared to that with C2-ceramide alone and induced pyknosis. Annexin V/7-aminoactinomycin D (7AAD) double staining and Western blotting indicated that apoptosis was involved in diTFPP/C2-ceramide cotreatment-mediated cell death. We next analyzed transcriptome alterations in diTFPP/C2-ceramide-cotreated HA22T cells with next-generation sequencing (NGS). The data indicated that diTFPP treatment disrupted sphingolipid metabolism, inhibited cell cycle-associated gene expression, and induced autophagy and reactive oxygen species (ROS)-responsive changes in gene expression. Additionally, we assessed the activation of autophagy with acridine orange (AO) staining and observed alterations in the expression of the autophagic proteins LC3B-II and Beclin-1, which indicated autophagy activation after diTFPP/C2-ceramide cotreatment. Elevated levels of ROS were also reported in diTFPP/C2-ceramide-treated cells, and the expression of the ROS-associated proteins SOD1, SOD2, and catalase was upregulated after diTFPP/C2-ceramide treatment. This study revealed the potential regulatory mechanism of the novel compound diTFPP in sphingolipid metabolism by showing that it disrupts ceramide metabolism and apoptotic sphingolipid accumulation. |
| 關鍵字： | ROS; apoptosis; autophagy; diTFPP; hepatocellular carcinoma (HCC); phenoxyphenol compound |
| 著作名稱： | LRWD1 expression is regulated through DNA methylation in human testicular embryonal carcinoma cells |
| 年度： | 2021 |
| 類別： |
期刊論文
Basic and clinical andrology.
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| 摘要： | Background: Sperm growth and maturation are correlated with the expression levels of Leucine-rich repeat and WD repeat-containing protein 1 (LRWD1), a widely expressed protein in the human testicles. The decrease in LRWD1 cellular level was linked to the reduction in cell growth and mitosis and the rise in cell microtubule atrophy rates. Since DNA methylation has a major regulatory role in gene expression, this study aimed at exploring the effect of the modulation of DNA methylation on LRWD1 expression levels.
Results: The results revealed the presence of a CpG island up of 298 bps (- 253 ~ + 45) upon LRWD1 promoter in NT2/D1 cells. The hypermethylation of the LRWD1 promoter was linked to a reduction in the transcription activity in NT2/D1 cells, as indicated by luciferase reporter assay. The methylation activator, floxuridine, confirmed the decrease in the LRWD1 promoter transcriptional activity. On the other hand, 5-Aza-2-deoxycytidine (5-Aza-dc, methylation inhibitor), significantly augmented LRWD1 promoter activity and the expression levels of mRNA and proteins. Furthermore, DNA methylation status of LRWD1 promoter in human sperm genomic DNA samples was analyzed. The results indicated that methylation of LRWD1 promoter was correlated to sperm activity.
Conclusions: Thus, the regulation of LRWD1 expression is correlated with the methylation status of LRWD1 promoter, which played a significant role in the modulation of spermatogenesis, sperm motility, and vitality. Based on these results, the methylation status of LRWD1 promoter may serve as a novel molecular diagnostic marker or a therapeutic target in males infertility.
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| 關鍵字： | 5-Aza-2′-deoxycytidine; DNA methylation; Floxuridine; LRWD1 |
| 著作名稱： | Etoposide Triggers Cellular Senescence by Inducing Multiple Centrosomes and Primary Cilia in Adrenocortical Tumor Cells |
| 年度： | 2021 |
| 類別： |
期刊論文
Cells
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| 摘要： | Etoposide (ETO) has been used in treating adrenocortical tumor (ACT) cells. Our previous study showed that ETO inhibits ACT cell growth. In the present study, we show that ETO treatment at IC50 (10 μM) inhibited ACT cell growth by inducing cellular senescence rather than apoptosis. Several markers of cellular senescence, including enlarged nuclei, activated senescence-associated β-galactosidase activity, elevated levels of p53 and p21, and down-regulation of Lamin B1, were observed. We further found that ETO induced multiple centrosomes. The inhibition of multiple centrosomes accomplished by treating cells with either roscovitine or centrinone or through the overexpression of NR5A1/SF-1 alleviated ETO-induced senescence, suggesting that ETO triggered senescence via multiple centrosomes. Primary cilia also played a role in ETO-induced senescence. In the mechanism, DNA-PK-Chk2 signaling was activated by ETO treatment; inhibition of this signaling cascade alleviated multiple ETO-induced centrosomes and primary cilia followed by reducing cellular senescence. In addition to DNA damage signaling, autophagy was also triggered by ETO treatment for centrosomal events and senescence. Importantly, the inactivation of DNA-PK-Chk2 signaling reduced ETO-triggered autophagy; however, the inhibition of autophagy did not affect DNA-PK-Chk2 activation. Thus, ETO activated the DNA-PK-Chk2 cascade to facilitate autophagy. The activated autophagy further induced multiple centrosomes and primary cilia followed by triggering senescence. |
| 關鍵字： | Chk2; DNA-PK; autophagy; centrosome; etoposide; primary cilia; senescence |
| 著作名稱： | The Long-Term DEHP Exposure Confers Multidrug Resistance of Triple-Negative Breast Cancer Cells through ABC Transporters and Intracellular ROS |
| 年度： | 2021 |
| 類別： |
期刊論文
Antioxidants (Basel)
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| 摘要： | The characteristics of phthalates had been thought to be similar to endocrine disruptors, which increases cancer risk. The role of phthalates in acquired drug resistance remains unclear. In this study, we investigated the effect of di-(2-ethylhexyl) phthalate (DEHP) on acquired drug resistance in breast cancer. MCF7 and MDA-MB-231 breast cancer cells were exposed to long-term physiological concentration of DEHP for more than three months. Long-exposure DEHP permanently attenuated the anti-proliferative effect of doxorubicin with estrogen receptor-independent activity even after withdrawal of DEHP. Long term DEHP exposure significantly reduced ROS (O2-) level in MDA-MB-231 cells while increased in MCF7 cells. ATP-binding cassette (ABC) transporters possess a widely recognized mechanism of drug resistance and are considered a target for drug therapy. Upregulation of ABC family proteins, ABCB-1 and ABCC-1 observed in DEHP-exposed clones compared to doxorubicin-resistant (DoxR) and parental MDA-MB-231 cells. A viability assay showed enhanced multidrug resistance in DEHP-exposed clones against Dox, topotecan, and irinotecan. Inhibition of ABC transporters with tariquidar, enhanced drug cytotoxicity through increased drug accumulation reversing acquired multidrug resistance in MDA-MB-231 breast cancer cells. Tariquidar enhanced Dox cytotoxicity by increasing intracellular ROS production leading to caspase-3 mediated apoptosis. Activation of PI3K/Akt signaling enhanced proliferation and growth of DEHP-exposed MDA-MB-231 cells. Overall, long-term DEHP exposure resulted in acquired multidrug resistance by upregulating ABCB-1 and ABCC1; apart from proliferation PI3K/Akt may be responsible for acquired drug resistance through ABC transporter upregulation. Targeting ABCB1 and ABCC1 with tariquidar may be a promising strategy for reversing the acquired multidrug resistance of triple-negative breast cancer cells. |
| 關鍵字： | ATP-binding transporter proteins; ROS; breast cancer; drug resistance; long DEHP exposure; tariquidar |
| 著作名稱： | Inflammation-related pyroptosis, a novel programmed cell death pathway, and its crosstalk with immune therapy in cancer treatment |
| 年度： | 2021 |
| 類別： |
期刊論文
Theranostics
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| 摘要： | In recent decades, chemotherapies targeting apoptosis have emerged and demonstrated remarkable achievements. However, emerging evidence has shown that chemoresistance is mediated by impairing or bypassing apoptotic cell death. Several novel types of programmed cell death, such as ferroptosis, necroptosis, and pyroptosis, have recently been reported to play significant roles in the modulation of cancer progression and are considered a promising strategy for cancer treatment. Thus, the switch between apoptosis and pyroptosis is also discussed. Cancer immunotherapy has gained increasing attention due to breakthroughs in immune checkpoint inhibitors; moreover, ferroptosis, necroptosis, and pyroptosis are highly correlated with the modulation of immunity in the tumor microenvironment. Compared with necroptosis and ferroptosis, pyroptosis is the primary mechanism for host defense and is crucial for bridging innate and adaptive immunity. Furthermore, recent evidence has demonstrated that pyroptosis exerts benefits on cancer immunotherapies, including immune checkpoint inhibitors (ICIs) and chimeric antigen receptor T-cell therapy (CAR-T). Hence, in this review, we elucidate the role of pyroptosis in cancer progression and the modulation of immunity. We also summarize the potential small molecules and nanomaterials that target pyroptotic cell death mechanisms and their therapeutic effects on cancer. |
| 關鍵字： | Nonapoptotic programmed cell death; cell death switch; immunotherapy; inflammasome; pyroptosis |
| 著作名稱： | The Role of Necroptosis in ROS-Mediated Cancer Therapies and Its Promising Applications |
| 年度： | 2020 |
| 類別： |
期刊論文
Cancers (Basel)
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| 摘要： | Over the past decades, promising therapies targeting different signaling pathways have emerged. Among these pathways, apoptosis has been well investigated and targeted to design diverse chemotherapies. However, some patients are chemoresistant to these therapies due to compromised apoptotic cell death. Hence, exploring alternative treatments aimed at different mechanisms of cell death seems to be a potential strategy for bypassing impaired apoptotic cell death. Emerging evidence has shown that necroptosis, a caspase-independent form of cell death with features between apoptosis and necrosis, can overcome the predicament of drug resistance. Furthermore, previous studies have also indicated that there is a close correlation between necroptosis and reactive oxygen species (ROS); both necroptosis and ROS play significant roles both under human physiological conditions such as the regulation of inflammation and in cancer biology. Several small molecules used in experiments and clinical practice eliminate cancer cells via the modulation of ROS and necroptosis. The molecular mechanisms of these promising therapies are discussed in detail in this review. |
| 關鍵字： | |
| 著作名稱： | Identification of SEPTIN12 as a novel target of the androgen and estrogen receptors in human testicular cells |
| 年度： | 2019 |
| 類別： |
期刊論文
Biochimie
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| 摘要： | SEPTIN12 (SEPT12) is a testis-enriched gene that is downregulated in the testis of infertile men with severe spermatogenic defects. While SEPT12 is involved in spermatogenic failure and sperm motility disorder, SEPT12 transcriptional regulation is still unknown. Here we report the promoter region of SEPT12 as a 245 bp segment upstream from the transcription start site. One androgen receptor (AR) and two of estrogen receptor α (ERα) binding sites were on this region were identified initially by Bioinformatics prediction and confirmed by chromatin immunoprecipitation assay. Truncated ERα or AR binding sites decreased the promoter activity, which indicated that the ERα and AR are essential for the SEPT12 promoter. On the other hand, the promoter activity was enhanced by the treatment with 17β-estradiol (E2) and 5α-dihydrotestosterone (5α-DHT). Thus, one androgen and two estrogen hormone responsive elements were in the promoter of SEPT12 gene to regulate the SEPT12 expression.
Two single nucleotide polymorphisms (SNPs), rs759992 T>C and rs3827527 C>T, were observed in the SEPT12 gene promoter region and were able to decrease the promoter activity. In conclusion, the current work identified the promoter of the human SEPT12 gene and provided key evidence about its transcriptional regulation via E2 and 5α-DHT. Since SEPT12 has an important role in spermatogenesis, SEPT12 expression analysis can be developed as a potential tool for the assessment of environmental or food pollution by hormones or for the evaluation of the risk of endocrine-disrupting chemicals (EDCs) in general.
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| 關鍵字： | SEPTIN12; androgen receptor; estrogen receptor α; core promoter; transcriptional regulation; SNPs |
| 著作名稱： | LRWD1 Regulates Microtubule Nucleation and Proper Cell Cycle Progression in the Human Testicular Embryonic Carcinoma Cells |
| 年度： | 2018 |
| 類別： |
期刊論文
Journal of cellular biochemistry
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| 摘要： | Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is a testis-specific protein that mainly expressed in the sperm neck where centrosome is located. By using microarray analysis, LRWD1 is identified as a putative gene that involved in spermatogenesis. However, its role in human male germ cell development has not been extensively studied. When checking in the semen of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia, the level of LRWD1 in the sperm neck was significantly reduced with a defective neck or tail. When checking the sub-cellular localization of LRWD1 in the cells, we found that LRWD1 resided in the centrosome and its centrosomal residency was independent of microtubule transportation in NT2/D1, the human testicular embryonic carcinoma, cell line. Depletion of LRWD1 did not induce centrosome re-duplication but inhibited microtubule nucleation. In addition, the G1 arrest were observed in LRWD1 deficient NT2/D1 cells. Upon LRWD1 depletion, the levels of cyclin E, A, and phosphorylated CDK2, were reduced. Overexpression of LRWD1 promoted cell proliferation in NT2/D1, HeLa, and 239T cell lines. In addition, we also observed that autophagy was activated in LRWD1 deficient cells and inhibition of autophagy by chloroquine or bafilomycin A1 promoted cell death when LRWD1 was depleted. Thus, we found a novel function of LRWD1 in controlling microtubule nucleation and cell cycle progression in the human testicular embryonic carcinoma cells. |
| 關鍵字： | LRWD1; CENTROSOME; CELL CYCLE; MICROTUBULE NUCLEATION; AUTOPHAGY |
| 著作名稱： | Nuclear factor erythroid-2-related factor regulates LRWD1 expression and cellular adaptation to oxidative stress in human embryonal carcinoma cells. |
| 年度： | 2018 |
| 類別： |
期刊論文
Biochimie
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| 摘要： | Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is implicated in the regulation of signal transduction, transcription, RNA processing and tumor development. However, LRWD1 transcriptional regulation is not fully understood. This study aimed to investigate the relationship between LRWD1 expression and reactive oxygen species (ROS) level in human embryonal carcinoma cell line, NT2/D1 cells, which will help in understanding the transcriptional regulatory role of ROS in cells. Results showed that the exposure of NT2/D1 cells to various concentrations of hydrogen peroxide (H2O2) and the nitric oxide (NO) donor sodium nitroprusside (SNP) caused a significant increase in the mRNA and protein expression of LRWD1. In addition, LRWD1 promoter luciferase reporter assay, and Chromatin Immunoprecipitation assay (CHIP assay) showed that nuclear factor erythroid-2-related factor (Nrf2) was involved in the regulation of LRWD1 expression in response to oxidative stress. The involvement of Nrf2 was confirmed by shRNA-mediated knockdown of Nrf2 in NT2/D1 cells, which caused a significant decrease in LRWD1 expression in response to oxidative stress. Similarly, LRWD1 knockdown resulted in the accumulation of H2O2 and superoxide anion radical (O2-). Blocking ROS production by N-acetyl cysteine (NAC) protected NT2/D1 shLRWD1cells from H2O2-induced cell death. Collectively, oxidative stress increased LRWD1 expression through a Nrf2-dependent mechanism, which plays an important role in cellular adaptation to oxidative stress. These results highlight an evidence, on the molecular level, about LRWD1 transcriptional regulation under oxidative stress. |
| 關鍵字： | LRWD1; Nrf2; Oxidative stress; Reactive oxygen species; Transcriptional regulation |
| 著作名稱： | Enhancing the Anticancer Activity of Antrodia cinnamomea in Hepatocellular Carcinoma Cells via Cocultivation With Ginger: The Impact on Cancer Cell Survival Pathways. |
| 年度： | 2018 |
| 類別： |
期刊論文
Frontiers in pharmacology
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| 摘要： | Antrodia cinnamomea (AC) is a medicinal fungal species that has been widely used traditionally in Taiwan for the treatment of diverse health-related conditions including cancer. It possesses potent anti-inflammatory and antioxidant properties in addition to its ability to promote cancer cell death in several human tumors. Our aim was to improve the anticancer activity of AC in hepatocellular carcinoma (HCC) through its cocultivation with ginger aiming at tuning the active ingredients. HCC cell lines, Huh-7 and HepG2 were used to study the in vitro anticancer activity of the ethanolic extracts of AC (EAC) alone or after the cocultivation in presence of ginger (EACG). The results indicated that the cocultivation of AC with ginger significantly induced the production of important triterpenoids and EACG was significantly more potent than EAC in targeting HCC cell lines. EACG effectively inhibited cancer cells growth via the induction of cell cycle arrest at G2/M phase and induction of apoptosis in Huh-7 and HepG2 cells as indicated by MTT assay, cell cycle analysis, Annexin V assay, and the activation of caspase-3. In addition, EACG modulated cyclin proteins expression and mitogen-activated protein kinase (MAPK) signaling pathways in favor of the inhibition of cancer cell survival. Taken together, the current study highlights an evidence that EACG is superior to EAC in targeting cancer cell survival and inducing apoptotic cell death in HCC. These findings support that EACG formula can serve as a potential candidate for HCC adjuvant therapy. |
| 關鍵字： | Antrodia cinnamomea; MAPK; adjuvant therapy; apoptosis; cell cycle; ginger; hepatocellular carcinoma |
| 著作名稱： | Exogenous C₈-Ceramide Induces Apoptosis by Overproduction of ROS and the Switch of Superoxide Dismutases SOD1 to SOD2 in Human Lung Cancer Cells |
| 年度： | 2018 |
| 類別： |
期刊論文
International journal of molecular sciences
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| 摘要： | Ceramides, abundant sphingolipids on the cell membrane, can act as signaling molecules to regulate cellular functions including cell viability. Exogenous ceramide has been shown to exert potent anti-proliferative effects against cancer cells, but little is known about how it affects reactive oxygen species (ROS) in lung cancer cells. In this study, we investigated the effect of N-octanoyl-D-erythro-sphingosine (C₈-ceramide) on human non-small-cell lung cancer H1299 cells. Flow cytometry-based assays indicated that C₈-ceramide increased the level of endogenous ROS in H1299 cells. Interestingly, the ratio of superoxide dismutases (SODs) SOD1 and SOD2 seem to be regulated by C₈-ceramide treatment. Furthermore, the accumulation of cell cycle G1 phase and apoptotic populations in C₈-ceramide-treated H1299 cells was observed. The results of the Western blot showed that C₈-ceramide causes a dramatically increased protein level of cyclin D1, a critical regulator of cell cycle G1/S transition. These results suggest that C₈-ceramide acts as a potent chemotherapeutic agent and may increase the endogenous ROS level by regulating the switch of SOD1 and SOD2, causing the anti-proliferation, and consequently triggering the apoptosis of NSCLC H1299 cells. Accordingly, our works may give a promising strategy for lung cancer treatment in the future. |
| 關鍵字： | C8-ceramide; ROS; SOD switch; apoptosis; cyclin D1; lung cancer |
| 著作名稱： | Inhibitory effect of trans-ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability. |
| 年度： | 2016 |
| 類別： |
期刊論文
Chinese Medicine
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| 摘要： | BACKGROUND:
Trans-ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of trans-FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of trans-FA on the H1299 human lung cancer cell line.
METHODS:
The 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2,7-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boydens well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.
RESULTS:
DPPH assay results indicated that trans-FA exerted potent antioxidant effects. However, trans-FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. Trans-FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, trans-FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. Trans-FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, trans-FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.
CONCLUSION:
Various concentrations (0.06-0.6 mM) of trans-FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299. |
| 關鍵字： | |
| 著作名稱： | The adverse effects of low-dose exposure to Di(2-ethylhexyl) phthalate during adolescence on sperm function in adult rats |
| 年度： | 2016 |
| 類別： |
期刊論文
Environmental toxicology
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| 摘要： | Di(2-ethylhexyl) phthalate (DEHP) is the most crucial phthalate derivative added to polyvinyl chloride as a plasticizer. This study examined the effects of low-dose exposure to DEHP during adolescence on sperm function in adult rats. The male rats were daily gavaged with 30, 100, 300, and 1000 μg kg-1 of DEHP or corn oil from postnatal day (PND) 42 until PND 105. The selection of DEHP doses ranged from the mean daily intake by the normal-population exposure levels to no-observed-adverse-effect level of DEHP for the endpoints evaluated until adulthood. Significant increases in the percentage of sperm with tail abnormality, tendency for sperm DNA fragmentation index (DFI) and percentage of sperm with DFI were found in those exposed to 100, 300, and 1000 μg kg-1 (P??0.05). We observed a significant increase of hydrogen peroxide (H2 O2 ) generation in the sperm of the 1000 μg kg-1 group compared with the control group (P??0.05). The excessive production of sperm H2 O2 coincided with an increase in sperm DFI. In this study, the lowest-observed-adverse-effect level for sperm toxicity was considered to be 100 μg DEHP/kg/day in sperm morphology and chromatin DNA damage. Further research is necessary to clarify the mechanisms of DEHP-related sperm ROS generation on sperm DNA damage. c 2014 Wiley Periodicals, Inc. Environ Toxicol, 2014. |
| 關鍵字： | |
| 著作名稱： | Reactive oxygen species mediate Terbufos-induced apoptosis in mouse testicular cell lines via the modulation of cell cycle and pro-apoptotic proteins |
| 年度： | 2015 |
| 類別： |
期刊論文
Environmental toxicology
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| 摘要： | Terbufos (S-t-butylthiomethyl-O,O-diethyl phosphorodithioate) is a highly toxic organophosphate which is extensively used as an insecticide and nematicide. Chronic exposure to terbufos causes neuronal injury and predisposes to neurodegenerative diseases. Accumulating evidence has shown that the exposure to terbufos, as an occupational risk factor, may also cause reproductive disorders. However, the exact mechanisms of reproductive toxicity remain unclear. The present study aimed to investigate the toxic effect of terbufos on testicular cells and to explore the mechanism of toxicity on a cellular level. The cytotoxic effects of terbufos on mouse immortalized spermatogonia (GC-1), spermatocytes (GC-2), Leydig (TM3), and Sertoli (TM4) cell lines were assessed by MTT assays, caspase activation, flow cytometry, TUNEL assay, Western blot, and cell cycle analysis. The exposure to different concentrations of terbufos ranging from 50 to 800 μM for 6 h caused significant death in all the used testicular cell lines. Terbufos increased reactive oxygen species (ROS) production, reduced mitochondrial membrane potential, and initiated apoptosis, which was confirmed by a dose-dependent increase in the number of TUNEL-positive apoptotic cells. Blocking ROS production by N-acetyl cysteine (NAC) protected GC-1 cells from terbufos-induced cell death. The results demonstrated that terbufos induces ROS, apoptosis, and DNA damage in testicular cell lines and it should be considered potentially hazardous to testis. Together, this study provided potential molecular mechanisms of terbufos-induced toxicity in testicular cells and suggests a possible protective measure. © 2015 Wiley Periodicals, Inc. Environ Toxicol, 2015.
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| 關鍵字： | terbufos, mouse testicular cell lines, apoptosis, caspase-3, reactive oxygen species. |
| 著作名稱： | 6-Shogaol induces cell cycle arrest and apoptosis in human hepatoma cells through pleiotropic mechanisms |
| 年度： | 2015 |
| 類別： |
期刊論文
European journal of pharmacology
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| 摘要： | Abstract
Shogaols are a group of the active constituents of ginger that have been identified to have various biological activities. The aim of the current study was to investigate the antitumor activity of 6-shogaol in hepatocellular carcinoma (HCC) and the possible involvement of reactive oxygen species as a putative mechanism of action. HCC cell lines, HepG2 and Huh-7, were used to study the in vitro anti-cancer activity of 6-shogaol via the application of various molecular biology techniques. Results showed that 6-shogaol effectively inhibited the cell viability, caused cell cycle arrest at G2/M phase and induced apoptosis in HCC cells as indicated by MTT assay, DAPI nuclear staining, annexin V assay, cell cycle analysis, and activation of caspase-3. Western blot analysis revealed the ability of 6-shogaol to target cancer survival signaling pathways mediated by mitogen-activated protein kinase (MAPK), 5 AMP-activated protein kinase (AMPK) and Akt. In addition, 6-Shogaol induced alteration of cyclin proteins expression and caused cleavage of protein kinase C delta. Furthermore, 6-Shogaol was able to induce the production of reactive oxygen species and endoplasmic reticulum (ER) stress-associated proteins and the consequent activation of autophagy in HepG2 cells. Taken together, the current study highlights evidences that 6-shogaol induces apoptosis, modulates cyclins expression and targets cancer survival signaling pathways in HCC cell lines, at least in part, via the production of reactive oxygen species. These findings support 6-shogaols clinical promise as a potential candidate for HCC therapy.
Copyright © 2015 Elsevier B.V. All rights reserved. |
| 關鍵字： | 6-Shogaol; Apoptosis; Autophagy; Cell cycle arrest; Endoplasmic reticulum stress; Mitogen-activated protein; Reactive oxygen species |
| 著作名稱： | Correlation between leucine rich domain and the stability of LRWD1 protein in human NT2/D1 cells |
| 年度： | 2014 |
| 類別： |
期刊論文
Advances in medical sciences
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| 摘要： | PURPOSE: LRWD1 is a protein that contains LRR and WDs domains and is important in regulating spermatogenesis. However, the roles of LRR or WDs domains in the expression of LRWD1 remain unclear.
MATERIALS AND METHODS: The NT2/D1 cells separately transfected with full length of LRWD1 gene (LRWD(WT)) or genes with deleted sequences in the LRR domain (LRWD1(ΔLRR)), WD1 domain (LRWD1(ΔWD1)), WD2 domain (LRWD1(ΔWD2)), WD3 domain (LRWD1(ΔWD3)) and entire three WD domains (LRWD1(Δ3×WD)) were applied to investigate the expression levels of LRWD1 protein by either Western blot or flow cytometry. The associated proteins in these mutated LRWD1 proteins were identified by mass spectrometry.
RESULTS: Deletion of the LRR domain significantly decreased the expression of LRWD1 protein. With the treatment of MG132, the LRR domain may functions in preventing LRWD1 protein from proteasome-mediated degradation. In the co-immunoprecipitation analysis, protein receptor of tumor necrosis factor 2 (TNFR2) was specifically observed to be associated with LRR-deficient LRWD1 protein.
CONCLUSIONS: The LRR domain is significantly correlated to the stability of LRWD1 protein. Determining if the stability is modulated by TNFR2 is worthy of further study.
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| 關鍵字： | |
| 著作名稱： | The antiproliferative and apoptotic effects of sirtinol, a sirtuin inhibitor on human lung cancer cells by modulating Akt/β-catenin-Foxo3a axis |
| 年度： | 2014 |
| 類別： |
期刊論文
ScientificWorldJournal
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| 摘要： | Sirtuins, NAD(+)-dependent deacetylases, could target both histones and nonhistone proteins in mammalian cells. Sirt1 is the major sirtuin and has been shown to involve various cellular processes, including antiapoptosis, cellular senescence. Sirt1 was reported to be overexpressed in many cancers, including lung cancer. Sirtinol, a specific inhibitor of Sirt1, has been shown to induce apoptosis of cancer cells by elevating endogenous level of reactive oxygen species. In the study, we investigated the effect of sirtinol on the proliferation and apoptosis of nonsmall cell lung cancer (NSCLC) H1299 cells. The results of proliferation assay and colony formation assay showed the antigrowth effect of sirtinol. The annexin-V staining further confirmed the apoptosis induction by sirtinol treatment. Interestingly, the levels of phosphorylated Akt and β-catenin were significantly downregulated with treating the apoptotic inducing doses. On the contrary, sirtinol treatment causes the significantly increased level of FoxO3a, a proapoptotic transcription factor targeted by Sirt1. These above results suggested that sirtinol may inhibit cell proliferation of H1299 cells by regulating the axis of Akt-β-catenin-FoxO3a. Overall, this study demonstrates that sirtinol attenuates the proliferation and induces apoptosis of NSCLC cells, indicating the potential treatment against NSCLC cells by inhibiting Sirt1 in future applications. |
| 關鍵字： | |
| 著作名稱： | SEPT12-microtubule complexes are required for sperm head and tail formation |
| 年度： | 2013 |
| 類別： |
期刊論文
International journal of molecular sciences
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| 摘要： | SEPT12-microtubule complexes are required for sperm head and tail formation.
Kuo PL1, Chiang HS, Wang YY, Kuo YC, Chen MF, Yu IS, Teng YN, Lin SW, Lin YH.
The septin gene belongs to a highly conserved family of polymerizing GTP-binding cytoskeletal proteins. SEPTs perform cytoskeletal remodeling, cell polarity, mitosis, and vesicle trafficking by interacting with various cytoskeletons. Our previous studies have indicated that SEPTIN12+/+/+/- chimeras with a SEPTIN12 mutant allele were infertile. Spermatozoa from the vas deferens of chimeric mice indicated an abnormal sperm morphology, decreased sperm count, and immotile sperm. Mutations and genetic variants of SEPTIN12 in infertility cases also caused oligozoospermia and teratozoospermia. We suggest that a loss of SEPT12 affects the biological function of microtublin functions and causes spermiogenesis defects. In the cell model, SEPT12 interacts with α- and β-tubulins by co-immunoprecipitation (co-IP). To determine the precise localization and interactions between SEPT12 and α- and β-tubulins in vivo, we created SEPTIN12-transgene mice. We demonstrate how SEPT12 interacts and co-localizes with α- and β-tubulins during spermiogenesis in these mice. By using shRNA, the loss of SEPT12 transcripts disrupts α- and β-tubulin organization. In addition, losing or decreasing SEPT12 disturbs the morphogenesis of sperm heads and the elongation of sperm tails, the steps of which are coordinated and constructed by α- and β-tubulins, in SEPTIN12+/+/+/- chimeras. In this study, we discovered that the SEPTIN12-microtubule complexes are critical for sperm formation during spermiogenesis.
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| 關鍵字： | septin 12 |
| 著作名稱： | A single-nucleotide polymorphism of the DAZL gene promoter confers susceptibility to spermatogenic failure in the Taiwanese Han.Teng YN, Chang YP, Tseng JT, Kuo PH, Lee IW, Lee MS, Kuo PL. Human Reproduction, 27(9):2857-65. (SCI, IF 4.475, Ranking/Category 3/79 3.79% in OBSTETRICS & GYNECOLOGY). NSC 97-2628-B-006-012-MY3. |
| 年度： | 2012 |
| 類別： |
期刊論文
Hum Reprod
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| 摘要： | Hum Reprod. 2012 Sep;27(9):2857-65. Epub 2012 Jun 29.
A single-nucleotide polymorphism of the DAZL gene promoter confers susceptibility to spermatogenic failure in the Taiwanese Han.
Teng YN, Chang YP, Tseng JT, Kuo PH, Lee IW, Lee MS, Kuo PL.
SourceDepartment of Biological Sciences and Technology, National University of Tainan, Tainan, Taiwan.
Abstract
BACKGROUND Deleted in AZoospermia-like (DAZL) is an autosomal homologue of Y chromosome-linked DAZ gene located on chromosome 3p24. DAZL is only expressed in the gonads and is critical to germ cell development in different species. However, the regulation of DAZL has not been explored. METHODS Reporter assays, electrophoretic mobility shift assays, supershift assays and bisulfate sequencing were used to identify the core promoter region of DAZL. Sequence analysis was used to identify single-nucleotide polymorphisms (SNPs) in the promoter region. A total of 337 infertile men with abnormal semen parameters and 203 fertile men with normal semen parameters were subjected to sequence analysis of the DAZL promoter region. RESULTS The DAZL gene core promoter is located 1 kb upstream of the transcription start site. Three SNPs (-792G>A, -669A>C and -309T>C) were identified in our population. Of these three SNPs, -792G>A was more prevalent in the infertile men (P 0.0005). Quantitative analysis revealed that genotypes of -792G>A had effects on sperm concentration (P 0.0025) and motility (P 1.5 × 10(-7)). The G to A substitution was associated with decreased binding of the nuclear respiratory factor-1 (NRF-1) to the promoter region and decreased reporter gene activity. CONCLUSION We have identified the core promoter of the human DAZL gene. We also provide preliminary evidence for the role of a novel SNP of the DAZL gene promoter in human spermatogenic failure.
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| 關鍵字： | single-nucleotide polymorphism / DAZL / gene promoter / spermatogenic failure |
| 著作名稱： | Endoplasmic Reticulum Stress Stimulates p53 Expression through NF-κB Activation.Lin WC, Chuang YC, Chang YS, Lai MD, Teng YN, Su IJ, Wang CC, Lee KH, Hung JH. Plos One, 7(7):e39120. (SCI, IF 4.092, Ranking/Category 12/85 14.11% in BIOLOGY). NSC 99-2320-B-041-001. |
| 年度： | 2012 |
| 類別： |
期刊論文
PLoS One.
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| 摘要： | Abstract
BACKGROUND: Induction of apoptosis by endoplasmic reticulum (ER) stress is implicated as the major factor in the development of multiple diseases. ER stress also appears to be a potentially useful major response to many chemotherapeutic drugs and environmental chemical compounds. A previous study has indicated that one major apoptotic regulator, p53, is significantly increased in response to ER stress, and participates in ER stress-induced apoptosis. However, the regulators of p53 expression during ER stress are still not fully understood.
PRINCIPAL FINDINGS: In this report, we demonstrate that induction of p53 expression is mediated through NF-κB signaling pathways during ER stress in MCF-7 cells. Tunicamycin or brefeldin A, two ER stress inducers, increased p53 expression in MCF-7 and Hela cells. We found p53 nuclear localization, activity, and phosphorylation at serine 15 on p53 increased during ER stress. Nuclear translocation of NF-κB and activity of NF-κB were also observed during ER stress. ER stress-induced p53 expression was significantly inhibited by coincubation with the NF-κB inhibitor, Bay 11-7082 and downregulation of NF-κB p65 expression. The role of p53 in mediating Brefeldin A-induced apoptosis was also investigated. Induction of p53 expression by Brefeldin A was correlated to Brefeldin A-induced apoptosis. Furthermore, downregulation of p53 expression by p53 siRNA significantly reduced Brefeldin A-induced apoptosis in MCF-7 cells.
SIGNIFICANCE: Taken together, NF-κB activation and induction of p53 expression is essential for ER stress-induced cell death which is important for therapeutic effects of clinical cancer drugs. Our results may provide insight into the mechanism of cancer chemotherapy efficacy that is associated with induction of ER stress.
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| 關鍵字： | NF-kB, p53, Endoplasmic Reticulum Stress |
| 著作名稱： | Nuclear factor-κB (NF-κB) Regulates the Expression of Human Testis-Enriched Leucine-rich Repeats and WD repeat Domain containing 1 (LRWD1) Gene, Yen-Ni Teng*, Po-Jung Chuang, and Yo-Wen Liu. International Journal of Molecular Sciences 2012; 14(1):625-39. (SCI, IF 2.598, Ranking/Category 45/154 29.22% in CHEMISTRY, MULTIDISCIPLINARY). NSC 99-2314-B-024-001-MY3. |
| 年度： | 2012 |
| 類別： |
期刊論文
International Journal of Molecular Sciences, 2012
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| 摘要： | The human Leucine-rich Repeats and WD repeat Domain containing 1 (LRWD1) gene was originally identified by cDNA microarray as one of the genes down-regulated in the testicular tissues of patients with severe spermatogenic defects. Human LRWD1 is a testicular-enrich protein that is present predominantly in the cytoplasm of spermatocytes and spermatids and colocalizes with the centrosome at the base of sperm tail. Reporter assay, Chromatin immunoprecipitation (ChIP) analysis, and gel electrophoretic mobility shift assay (EMSA) were used to identify the core promoter region of LRWD1. A 198 bp segment upstream of the LRWD1 transcription initiation site exhibited promoter activity. The LRWD1 core promoter lacked a TATA box but contained a NF-B binding site. Chromatin immunoprecipitation (ChIP) analysis and gel electrophoretic mobility shift assay (EMSA) showed basal binding of the NF-κB subunit to the LRWD1 promoter. LRWD1 promoter activity was positively regulated by NF-κB, and this regulation was dependent on the presence of the conserved κB site in the LRWD1 promoter region. Our data suggest that NF-κB is an important regulator for the expression of LRWD1. This is the first study showing that the expression of the testis-enriched LRWD1 gene is regulated by NF-κB. |
| 關鍵字： | NF-κB; LRWD1; promoter |
| 著作名稱： | Induction of Bcl-2 expression by hepatitis B virus pre-S2 mutant large surface protein resistance to 5-fluorouracil treatment in Huh-7 cells. Hung JH, Teng YN, Wang LH, Su IJ, Wang CC, Huang W, Lee KH, Lu KY, Wang LH. Plos One, 6(12):e28977. (SCI, IF 4.092, Ranking/Category 12/85 14.11% in BIOLOGY). NSC 100-2320-B-041-007. |
| 年度： | 2011 |
| 類別： |
期刊論文
PLoS One
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| 摘要： | PLoS One. 2011;6(12):e28977. Epub 2011 Dec 22.
Induction of Bcl-2 expression by hepatitis B virus pre-S2 mutant large surface protein resistance to 5-fluorouracil treatment in Huh-7 cells.
Hung JH, Teng YN, Wang LH, Su IJ, Wang CC, Huang W, Lee KH, Lu KY, Wang LH.
SourceDepartment of Biotechnology, Chia Nan University of Pharmacy and Science, Tainan, Taiwan. hung86@mail.chna.edu.tw
Abstract
BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with poor prognosis due to resistance to conventional chemotherapy and limited efficacy of radiotherapy. Our previous studies have indicated that expression of Hepatitis B virus pre-S2 large mutant surface antigen (HBV pre-S2Δ) is associated with a significant risk of developing HCC. However, the relationship between HBV pre-S2Δ protein and the resistance of chemotherapeutic drug treatment is still unclear.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that the expression of HBV pre-S2Δ mutant surface protein in Huh-7 cell significantly promoted cell growth and colony formation. Furthermore, HBV pre-S2Δ protein increased both mRNA (2.7±0.5-fold vs. vehicle, p0.05) and protein (3.2±0.3-fold vs. vehicle, p0.01) levels of Bcl-2 in Huh-7 cells. HBV pre-S2Δ protein also enhances Bcl-2 family, Bcl-xL and Mcl-1, expression in Huh-7 cells. Meanwhile, induction of NF-κB p65, ERK, and Akt phosphorylation, and GRP78 expression, an unfolded protein response chaperone, were observed in HBV pre-S2Δ and HBV pre-S-expressing cells. Induction of Bcl-2 expression by HBV pre-S2Δ protein resulted in resistance to 5-fluorouracil treatment in colony formation, caspase-3 assay, and cell apoptosis, and can enhance cell death by co-incubation with Bcl-2 inhibitor. Similarly, transgenic mice showed higher expression of Bcl-2 in liver tissue expressing HBV pre-S2Δ large surface protein in vivo.
CONCLUSION/SIGNIFICANCE: Our result demonstrates that HBV pre-S2Δ increased Bcl-2 expression which plays an important role in resistance to 5-fluorouracil-caused cell death. Therefore, these data provide an important chemotherapeutic strategy in HBV pre-S2Δ-associated tumor.
c 2011 Hung et al.
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| 關鍵字： | HBV, Bcl-2, NF-kB |
| 著作名稱： | Expression of LRWD1 in mouse testis and its centrosomal localization. International Journal of Andrology, 33(6): 832-40. (SCI, IF 3.591, Ranking/Category 1/6 16.66% in ANDROLOGY). NSC 95-2320-B-041-006. |
| 年度： | 2010 |
| 類別： |
期刊論文
International Journal of Andrology
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| 摘要： | Abstract
The mouse leucine-rich repeats and WD repeat domain containing 1 (lrwd1) gene is located on chromosome 5qG2 and spans over 13 kilobases. It encodes a novel protein of 648-amino acid protein that shares 78.3% amino acid sequence identity with the human LRWD1 protein. We used an oligopeptide as immunogen to generate an anti-lrwd1 antibody in rabbits. Both Northern and Western blot results indicated that the expression of lrwd1 is testis specific. Immunostaining of mouse testis sections detected high levels of lrwd1 signals in the cytoplasm of primary spermatocytes to mature spermatozoa and much weaker signals in spermatogonia. On mature spermatozoa, the anti-lrwd1 antibody stained strongly the connection region between the head and the neck where the centrosome is located. Additional immunostaining and immunoprecipitation showed colocalization and interaction between lrwd1 and γ-tubulin respectively, implicating lrwd1 as a candidate centrosomal protein. These results suggest that lrwd1 may play an important role in spermatogenesis.
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| 關鍵字： | LRWD1, centrosome, spermatogenesis, testis |
| 著作名稱： | Teng YN＊, Kuo PL, Cheng TC, Liao MH. Histone gene expression profile during Spermatogenesis. Fertility & Sterility 2010 93(7):2447-9. (SCI, IF4.167, Ranking 3/61 4.9% in OBSTETRICS & GYNECOLOGY) |
| 年度： | 2010 |
| 類別： |
期刊論文
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| 摘要： | Gene bank search and reverse transcription-polymerase chain reaction were used to analyze the expression profile for histone genes during spermatogenesis. The objective of this study was to provide a systematic view of histone gene expression.
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| 關鍵字： | histone, spermatogenesis, fertility |
| 著作名稱： | Pan HA, Lee YC, Teng YN, Tsai SJ, Lin YM, Kuo PL. CDC25 protein expression and interaction with DAZL in human corpus luteum. Fertility & Sterility 2009 92(6):1997-2003 (SCI, IF4.167, Ranking 3/61 4.9% in OBSTETRICS & GYNECOLOGY) |
| 年度： | 2009 |
| 類別： |
期刊論文
Fertility & Sterility
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| 摘要： | OBJECTIVE: To determine whether the human corpus luteum exhibits CDC25 protein expression and its expression pattern, and identify the interaction with DAZL (deleted in azoospermia-like) in human luteal cells.
DESIGN: Experimental study.
SETTING: Medical research laboratory in a university hospital.
PATIENT(S): Twelve human corpus luteum (CL), four in the early stage, four in the midstage, and four in the late stage of the luteal phase, respectively, and 10 granulosa-lutein cells from IVF patients.
INTERVENTION(S): Immunohistochemical stain and Western blot were used to characterize the expression of CDC25 protein, and protein immunoprecipitation was used to identify the interaction of CDC25 with DAZL in human luteal cells.
MAIN OUTCOME MEASURE(S): CDC25 protein expression pattern in different stages of luteal phase and interaction with DAZL.
RESULT(S): In this study, we show evidence that CDC25 protein is express in granulosa-lutein cells from different stages of CL, and identified only the CDC25A interaction with DAZL in luteal cells.
CONCLUSION(S): The CL is the final form of a developing follicle and is the major endocrine component of the ovary in maintaining early successful pregnancy. Expression of CDC25 protein throughout different stages of the ovarian cycles implies important functional roles in the regulation of female reproduction.
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| 關鍵字： | CDC25, corpus luteum, DAZL, luteal cell |
| 著作名稱： | Lee IW, Kuan LC, Lin CH, Pan HA, Hsu CC, Tsai YC, Kuo PL, Teng YN＊. Association of USP26 haplotypes in men in Taiwan, China with severe spermatogenic defect. Asian journal of andrology 2008 10(6):896-904. |
| 年度： | 2008 |
| 類別： |
期刊論文
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| 摘要： | AIM: To complete comprehensive haplotype analysis of USP26 for both fertile and infertile men.
METHODS: Two hundred infertile men with severe oligospermia or non-obstructive azoospermia were subjected to sequence analysis for the entire coding sequences of the USP26 gene. Two hundred men with proven fertility were genotyped by primer extension methods. Allele/genotype frequencies, linkage disequilibrium (LD) characteristics and haplotypes of fertile men were compared with infertile men.
RESULTS: The allele frequencies of five single nucleotide polymorphisms (370-371insACA, 494T>C, 576G>A, ss6202791C>T, 1737G>A) were significantly higher in infertile patients than control subjects. The major haplotypes in infertile men were TACCGA (28% of the population), TGCCGA (15%), TACCAA (8%), TGCCAA (6%), TATCAA (5%) and CATCAA (5%). The major haplotypes for the control subjects were TACCGA (58% of the population), CACCGA (7%), CATCGA (6%) and TGCCGA (5%). Haplotypes TGCCGA, TATCAA, CATCAA, CATCGC, TACCAA and TGCCAA were over-transmitted in patients with spermatogenic defect, whereas haplotypes TACCGA, CACCGA, and CATCGA were under-transmitted in these patients.
CONCLUSION: Some USP26 alleles and haplotypes are associated with spermatogenic defect in the Han nationality in Taiwan, China.
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| 關鍵字： | |
| 著作名稱： | Association of extremely skewed X-chromosome inactivation with Taiwanese women presenting with recurrent pregnancy loss.Kuo PL, Huang SC, Chang LW, Lin CH, Tsai WH, Teng YN.
J Formos Med Assoc. 2008 Apr;107(4):340-3.
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| 年度： | 2008 |
| 類別： |
期刊論文
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| 摘要： | X-chromosome inactivation (XCI) is a phenomenon that occurs in female mammals. Typically, maternally and paternally-derived X chromosomes are inactivated at approximately the same frequency. If preferential inactivation occurs, the person is considered to have skewed XCI. Skewed XCI has been reported to occur more frequently in women who experience recurrent pregnancy loss (RPL). In this study, we sought to investigate if there is an association between skewed XCI and unexplained RPL in Taiwanese women. A total of 194 women who had experienced unexplained RPL were recruited into the study. Human androgen receptor or DXS6673E and DX15-134 loci were used in the XCI assay. The results of our study suggested that a cut-off point less than 90% may not be justified for skewed XCI. Only extremely skewed (more than 95%) XCI is associated with RPL. Extremely skewed XCI occurs in a subset of Taiwanese women with RPL.
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| 關鍵字： | |
| 著作名稱： |
DAZL protein expression in mouse preimplantation embryo.
Pan HA, Liao RW, Chung CL, Teng YN, Lin YM, Kuo PL.
Fertil Steril. 2008 May;89(5 Suppl):1324-7. Epub 2007 Aug 30
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| 年度： | 2008 |
| 類別： |
期刊論文
|
| 摘要： | Abstract
OBJECTIVE: To evaluate the expression pattern of Dazl (deleted in azoospermia-like) protein in the mouse preimplantation embryo.
DESIGN: Experimental study.
SETTING: Medical research laboratory in a university hospital.
ANIMAL(S): Twenty female 28- to 35-day-old FVB mice.
INTERVENTION(S): Embryo collection at 1.5, 2.5, and 3.5 days postcoitus (plug date, 0.5 d postcoitus) to examine the Dazl protein expression from the two-cell embryo to the blastocyst.
MAIN OUTCOME MEASURE(S): Dazl protein expression was analyzed by immunofluorescent staining.
RESULT(S): There is abundant expression of Dazl protein in the cytoplasm of the blastomere. Strong fluorescent signals of Dazl protein expression were found in preimplantation embryo cytoplasm, including two-cell, eight-cell, morula, and blastocyst.
CONCLUSION(S): By using an antibody raised against mouse Daz-like protein (Dazl), we showed that Dazl protein is present in all cleaving stages of the preimplantation embryo. This is the first report on the protein expression of a Dazl gene during embryogenesis in mice. However, further study is needed to evaluate the molecular functional role of Dazl
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| 關鍵字： | |
| 著作名稱： | Screening of Prader-Willi syndrome and Angelman syndrome in school children with moderate to profound mental retardation in southern Taiwan.
Su MT, Teng YN, Kuo PL.
Acta Paediatr Taiwan. 2007 Mar-Apr;48(2):73-6.
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| 年度： | 2007 |
| 類別： |
期刊論文
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| 摘要： | BACKGROUND AND PURPOSE: Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by deficiencies of gene expression from paternal or maternal chromosome 15q11-q13, respectively. The study was conducted to estimate the prevalence of PWS and AS in children with moderate to profound mental retardation in Taiwan.
METHODS: The screening began with methylation studies in all enrolled cases. If methylation results were positive, Fluorescence in situ hybridization (FISH) and polymerase chain reaction (PCR) was used to determine whether deletion, uniparental disomy, or imprinting mutation was present.
RESULTS: Of 1053 children with moderate to profound mental retardation, we identified three cases of AS (0.28%) and one case of PWS (0.09%).
CONCLUSIONS: The prevalence of PWS is lower than AS in school children with moderate to profound mental retardation. The greater number of AS identified than that of PWS is most likely a reflection of more severe mental retardation for AS than for PWS.
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| 關鍵字： | |
| 著作名稱： | Expression of various CDC25B isoforms in human spermatozoa.Teng YN, Chung CL, Lin YM, Pan HA, Liao RW, Kuo PL.Fertil Steril. 2007 Aug;88(2):379-82. Epub 2007 Mar 6.
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| 年度： | 2007 |
| 類別： |
期刊論文
Fertil Steril
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| 摘要： | OBJECTIVE: To explore the role of CDC25B protein in postmeiotic germ cells.
DESIGN: In vitro experiment.
SETTING: University-based reproductive genetics laboratory.
PATIENT(S): Fertile and infertile volunteers.
INTERVENTION(S): Reverse transcription-polymerase chain reaction (RT-PCR), real-time RT-PCR, and immunostaining for CDC25B.
MAIN OUTCOME MEASURE: Expression profiling of CDC25B in human spermatozoa.
RESULT(S): Four splicing variants (CDC25B1, B2, B3, and B4) are expressed in human spermatozoa. Immunofluorescence staining showed strong homogeneous staining in the midpiece of spermatozoa, and weak staining in the principal piece and cytosol of the head. The messenger RNA (mRNA) transcript level of CDC25B was increased in sperm samples of men with asthenospermia.
CONCLUSION(S): The expression of CDC25B in different cellular compartments of human spermatozoa suggests that there are diverse noncell-cycle-related functions of CDC25B in terminally differentiated human germ cells.
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| 關鍵字： | |
| 著作名稱： | A simplified gene-specific screen for Y chromosome deletions in infertile men.Teng YN, Lin YH, Tsai YC, Hsu CC, Kuo PL, Lin YM.Fertil Steril. 2007 Jun;87(6):1291-300. Epub 2007 Feb 12.
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| 年度： | 2007 |
| 類別： |
期刊論文
Fertil Steril
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| 摘要： | OBJECTIVE: To test the diagnostic efficiency of a gene-specific, five-marker screening strategy for the detection of Y chromosome deletions.
DESIGN: Prospective case study.
SETTING: University genetics laboratory and reproductive clinics.
PATIENT(S): Six hundred twenty-seven infertile men and 212 fertile men.
INTERVENTION(S): Peripheral blood samples were screened for Y chromosome deletions in a triple-blind fashion using three protocols: protocol I consisted of five gene-specific markers, including USP9Y, DBY, SMCY, RBM1, and DAZ; protocol II included 14 gene-specific markers; and protocol III consisted of six sequence-tagged sites (STSs) markers recommended by EAA/EMQN.
MAIN OUTCOME MEASURE(S): Deletion status of Y chromosome genes or sequence-tagged sites.
RESULT(S): Protocols I and II identified the same 41 infertile patients with Y deletions. Protocol III identified 38 infertile patients with Y deletions, and all 38 patients were also identified by protocols I and II. One patient with isolated USP9Y deletion and two patients with isolated DBY deletions, as detected by protocols I and II, could not be identified by protocol III.
CONCLUSION(S): We observed mostly consistent results between our protocols and the EAA/EMQN protocol. This gene-specific, five-marker screening panel provides the same diagnostic efficiency as the EAA/EMQN protocol and may be considered an alternative to the EAA/EMQN protocol.
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| 關鍵字： | |
| 著作名稱： | Identification of ten novel genes involved in human spermatogenesis by microarray analysis of testicular tissue.Lin YH, Lin YM, Teng YN, Hsieh TY, Lin YS, Kuo PL.Fertil Steril. 2006 Dec;86(6):1650-8. Epub 2006 Oct 30.
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| 年度： | 2006 |
| 類別： |
期刊論文
Fertil Steril
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| 摘要： | OBJECTIVE: To identify novel genes that are down-regulated in the testicular tissue of infertile men.
DESIGN: Prospective study.
SETTING: University-based reproductive clinics and genetics laboratory.
PATIENTS: Nine patients with normal spermatogenesis, and 15 patients with maturation arrest (MA) or Sertoli cell-only syndrome (SCOS).
INTERVENTION: Testicular samples of patients with the same histology were pooled for complementary DNA (cDNA) microarray analysis.
MAIN OUTCOME MEASURE: Novel, down-regulated genes.
RESULTS: In total, 300 genes were significantly down-regulated in SCOS or MA samples, and 10 novel sterility-related genes were identified. Of the 10 novel genes, 6 genes (Hs.126780, Hs.553658, Hs.274135, Hs.268122, Hs.531701, and Hs.171130) encode proteins with predictable functional domains, and all these functional domains are believed to correlate with spermatogenesis and/or spermiogenesis. Conversely, the other 4 genes (Hs.351582, Hs.407480, Hs.552781, and Hs.355570) do not encompass known functional domains. Two genes (Hs.407480 and Hs.552781) lack mouse orthologues. Most novel genes showed a testis-specific expression pattern in both mice and humans. Reverse transcription-polymerase chain reaction (RT-PCR) showed three distinct types of developmental stage-dependent expressions of message ribonucleic acid (mRNA) for these novel genes in murine testes.
CONCLUSION: These 10 novel genes provide targets to elucidate novel pathways involved in human spermatogenesis.
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| 關鍵字： | |
| 著作名稱： | Association of DAZL haplotypes with spermatogenic failure in infertile men.Teng YN, Lin YM, Sun HF, Hsu PY, Chung CL, Kuo PL.Fertil Steril. 2006 Jul;86(1):129-35. Epub 2006 May 30.
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| 年度： | 2006 |
| 類別： |
期刊論文
Fertil Steril
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| 摘要： | OBJECTIVE: To identify novel DAZL single nucleotide polymorphisms (SNPs) and to compare allele/genotype frequencies, linkage disequilibrium (LD) characteristics, and DAZL haplotypes between fertile and infertile men.
DESIGN: Prospective case study.
SETTING: University genetics laboratory and reproductive clinics.
PATIENT(S): Two hundred thirty-one infertile men and 191 men with proven fertility.
INTERVENTION(S): Single strand conformation polymorphism and sequence analysis for DAZL gene polymorphism screening were done for all subjects enrolled.
MAIN OUTCOME MEASURE(S): Novel SNPs, allele/genotype frequencies, LD characteristics, and DAZL haplotypes between fertile and infertile men.
RESULT(S): Five SNPs were identified: 260A>G, 386A>G, 520+34c>a, 584+28c>t and 796+36g>a. SNP 386A>G was significantly associated with spermatogenic failure and was mainly heterozygous in infertile patients. The major haplotypes in infertile men were AACTA (45.8%), followed by AACCG, AAATA, AAACG, and GGACG for 260A>G/386A>G/520+34c>a/584+28c>t/796+36g>a. The major haplotypes for the control subjects were AACCG (41.7 %), AAATA, and AACTA. Of all haplotypes, five showed significant differences in frequency between infertile men and control subjects. Haplotypes AACTA, AAACG, and GGACG were overtransmitted in patients with spermatogenic failure, whereas haplotypes AACCG and AAATA were undertransmitted in these patients.
CONCLUSION(S): Our study suggests the association of autosomal DAZL haplotypes with human spermatogenic failure.
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| 關鍵字： | |
| 著作名稱： | Association of spermatogenic failure with decreased CDC25A expression in infertile men.Cheng YS, Kuo PL, Teng YN, Kuo TY, Chung CL, Lin YH, Liao RW, Lin JS, Lin YM.Hum Reprod. 2006 Sep;21(9):2346-52. Epub 2006 May 23.
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| 年度： | 2006 |
| 類別： |
期刊論文
Hum Reprod
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| 摘要： | BACKGROUND: DAZ gene family is crucial for human spermatogenesis that requires the precise co-ordination of cell cycle events. CDC25A is recognized as the downstream substrate of DAZ gene family and is thought to function on the M-phase regulation of cell cycles. We investigated the expression profiles of CDC25A in the testes of infertile men and evaluated the relationship between CDC25A levels and testicular phenotype, clinical hormonal parameters and sperm retrieval results.
METHODS: The protein and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemistry and quantitative real-time-PCR. CDC25A in human spermatozoa was investigated by western blotting and immunofluorescence staining.
RESULTS: The CDC25A protein was expressed mainly in spermatocyte, spermatid and spermatozoa. CDC25A transcript levels were significantly decreased (P 0.0009) in patients with spermatogenic failure, especially in men with meiotic arrest and Sertoli cell-only syndrome. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval (P 0.005).
CONCLUSIONS: Decreased CDC25A is associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies are necessary to explore the functional roles of CDC25A in human spermatozoa.
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| 關鍵字： | |
| 著作名稱： | Uniform deletion junctions of complete azoospermia factor region c deletion in infertile men in Taiwan.Hsu CC, Kuo PL, Chuang L, Lin YH, Teng YN, Lin YM.Asian J Androl. 2006 Mar;8(2):205-11.
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| 年度： | 2006 |
| 類別： |
期刊論文
Asian J Androl.
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| 摘要： | AIM: To determine the deletion junctions of infertile men in Taiwan with azoospermia factor region c (AZFc) deletions and to evaluate the genotype/phenotype correlation.
METHODS: Genomic DNAs from 460 infertile men were examined. Bacterial artificial chromosome clones were used to verify the accuracy of polymerase chain reaction. Deletion junctions of the AZFc region were determined by analysis of sequence-tagged sites and gene-specific markers.
RESULTS: Complete AZFc deletions, including BPY2, CDY1 and DAZ genes, were identified in 24 men. The proximal breakpoints were clustered between sY1197 and sY1192, and the distal breakpoints were clustered between sY1054 and sY1125 in all but one of the 24 men. The testicular phenotypes of men with complete AZFc deletion varied from oligozoospermia, to hypospermatogenesis, to maturation arrest.
CONCLUSION: We identified a group of infertile men with uniform deletion junctions of AZFc in the Taiwan population. Despite this homogeneous genetic defect in the AZFc region, no clear genotype/phenotype correlation could be demonstrated.
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| 關鍵字： | |
| 著作名稱： | Decreased mRNA transcripts of M-phase promoting factor and its regulators in the testes of infertile men.Lin YM, Teng YN, Chung CL, Tsai WC, Lin YH, Lin JS, Kuo PL.Hum Reprod. Jan;21(1):138-44. Epub 2005 Sep 9.
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| 年度： | 2006 |
| 類別： |
期刊論文
Hum Reprod
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| 摘要： | BACKGROUND: M-phase promoting factor (MPF), which is comprised of Cyclin B and a catalytic subunit, Cdc2, is a key enzyme required for cells to enter M phase in both mitosis and meiosis. MPF activity is controlled by the stimulatory dephosphorylation of the Cdc25 family and the inhibitory phosphorylation of Wee1. We determined the levels of mRNA transcripts of MPF and its regulators in the testes of infertile men, and evaluated the relationship between the transcript levels and patients testicular phenotypes and sperm retrieval results.
METHODS AND RESULTS: The mRNA transcript levels of CDC2, CCNB1, CCNB2, CDC25A, CDC25B, CDC25C and WEE1 in the testes of 37 azoospermic patients were examined by quantitative real-time polymerase chain reaction. Significant decreases in CDC2, CCNB1, CCNB2, CDC25A, CDC25C and WEE1 mRNA transcript levels were detected in patients with spermatogenic failure. CDC2 mRNA transcript levels correlated significantly with those of CCNB1 and CCNB2 mRNA. Significantly higher CDC2, CCNB1, CCNB2, CDC25C and WEE1 mRNA transcript levels were detected in 18 patients with successful sperm retrieval than in 11 patients with failed sperm retrieval.
CONCLUSIONS: We suggest that the decreased mRNA transcripts of MPF and its regulators play important roles in human spermatogenesis
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| 關鍵字： | |
| 著作名稱： | Isochromosome of Yp in a man with Sertoli-cell-only syndrome.Lin YH, Chuang L, Lin YM, Lin YH, Teng YN, Kuo PL.Fertil Steril. 2005 Mar;83(3):764-6.
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| 年度： | 2005 |
| 類別： |
期刊論文
Fertil Steril
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| 摘要： | OBJECTIVE: To address phenotype/genotype correlation in a man with i(Y)(p10).
DESIGN: Case report.
SETTING: University-based reproductive genetics laboratory.
PATIENT(S): A 27-year-old azoospermic man with i(Y)(p10), relatively normal stature, and testicular Sertoli-cell-only syndrome.
INTERVENTION(S): Testicular biopsy, cytogenetic study, Y-chromosome deletion mapping analysis, fluorescence in situ hybridization (FISH).
MAIN OUTCOME MEASURE(S): Expression of Y-chromosome genes.
RESULT(S): We have identified one azoospermic man with i(Y)(p10) of 312 Taiwanese men presenting with a severe spermatogenic defect. Y-chromosome deletion mapping analysis confirmed deletions of all Yq sequences, including a putative growth controlling gene. Fluorescence in situ hybridization (FISH) analysis showed duplication of Yp material. The patient had normal stature considering midparental height. He also had no germ cells in the testicular tissue (Sertoli-cell-only syndrome) resulting from the loss of azoospermia factor in Yq.
CONCLUSION(S): Among structural rearrangements of the Y-chromosome, the isochromosome of Yp occurs very rarely. This case is the first reported case of an isochromosome Yp with a detailed description of testicular histology and body height.
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| 關鍵字： | |
| 著作名稱： | Messenger RNA transcripts of the meiotic regulator BOULE in the testis of azoospermic men and their application in predicting the success of sperm retrieval.Lin YM, Kuo PL, Lin YH, Teng YN, Nan Lin JS.Hum Reprod. 2005 Mar;20(3):782-8. Epub 2004 Dec 9.
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| 年度： | 2005 |
| 類別： |
期刊論文
Hum Reprod
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| 摘要： | BACKGROUND: Testicular sperm retrieval can lead to paternity for azoospermic patients with spermatogenic failure. The human BOULE gene, a meiotic regulator of germ cells, is a gene whose altered expression may be associated with sterility. We determined the levels of BOULE transcripts in the testes of azoospermic patients, and evaluated the relationship between BOULE transcript levels and patients testicular phenotypes, clinical parameters and sperm retrieval results. METHODS and
RESULTS: BOULE transcript levels in the testes of 41 azoospermic patients were examined by quantitative competitive-reverse transcription-polymerase chain reaction. A significant decrease in BOULE transcript levels was detected in patients with spermatogenic failure, and BOULE transcript levels progressively decreased with increasing severity of testicular failure. BOULE transcript levels did not correlate with the serum hormone parameters measured. Significantly higher BOULE transcript levels were detected in 19 patients with successful sperm retrieval than in 12 patients with failed sperm retrieval. When using a cut-off value of 0.5 for BOULE transcript ratio to predict the success of sperm retrieval, both the sensitivity and specificity value were 100%.
CONCLUSIONS: We suggest the BOULE transcript plays an important role in human spermatogenesis and that the levels may predict the presence of testicular sperm in patients with spermatogenic failure.
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| 關鍵字： | |
| 著作名稱： | diTFPP, a Phenoxyphenol, Sensitizes Hepatocellular Carcinoma Cells to C2-Ceramide-Induced Autophagic Stress by Increasing Oxidative Stress and ER Stress Accompanied by LAMP2 Hypoglycosylation |
| 年度： | 2022 |
| 類別： |
期刊論文
Cancers (Basel)
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| 摘要： | Hepatocellular carcinoma (HCC), the most common type of liver cancer, is the leading cause of cancer-related mortality worldwide. Chemotherapy is the major treatment modality for advanced or unresectable HCC; unfortunately, chemoresistance results in a poor prognosis for HCC patients. Exogenous ceramide, a sphingolipid, has been well documented to exert anticancer effects. However, recent reports suggest that sphingolipid metabolism in ceramide-resistant cancer cells favors the conversion of exogenous ceramides to prosurvival sphingolipids, conferring ceramide resistance to cancer cells. However, the mechanism underlying ceramide resistance remains unclear. We previously demonstrated that diTFPP, a novel phenoxyphenol compound, enhances the anti-HCC effect of C2-ceramide. Here, we further clarified that treatment with C2-ceramide alone increases the protein level of CERS2, which modulates sphingolipid metabolism to favor the conversion of C2-ceramide to prosurvival sphingolipids in HCC cells, thus activating the unfolded protein response (UPR), which further initiates autophagy and the reversible senescence-like phenotype (SLP), ultimately contributing to C2-ceramide resistance in these cells. However, cotreatment with diTFPP and ceramide downregulated the protein level of CERS2 and increased oxidative and endoplasmic reticulum (ER) stress. Furthermore, insufficient LAMP2 glycosylation induced by diTFPP/ceramide cotreatment may cause the failure of autophagosome-lysosome fusion, eventually lowering the threshold for triggering cell death in response to C2-ceramide. Our study may shed light on the mechanism of ceramide resistance and help in the development of adjuvants for ceramide-based cancer therapeutics. |
| 關鍵字： | LAMP2 glycosylation; autophagic stress; ceramide; diTFPP; endoplasmic reticulum (ER) stress; hepatocellular carcinoma (HCC); oxidative stress; phenoxyphenol compound; sphingolipid metabolism. |
| 著作名稱： | MicroRNA-320a enhances LRWD1 expression through the AGO2/FXR1-dependent pathway to affect cell behaviors and the oxidative stress response in human testicular embryonic carcinoma cells |
| 年度： | 2023 |
| 類別： |
期刊論文
Aging
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| 摘要： | Background: Testicular cancer is fairly rare and can affect fertility in adult male. Leucine-rich repeats and WD repeat domain-containing protein 1 (LRWD1) is a sperm-specific marker, which mainly affects sperm motility in reproduction. Our previous study demonstrated the impact of LRWD1 in testicular cancer development, however, the underlying mechanisms remain unclear.
Methods: In this study, various plasmids associated with LRWD1 and miR-320a manipulation were used to explore their roles and regulatory effect in NT2D1 cellular process. Dual-Glo luciferin luciferase system was used for LRWD1 transcriptional activity and qRT-PCR and western blot were used to determine gene and protein expression.
Results: The results suggest that miR-320a positively regulated LRWD1 and positively correlated with proliferation of NT2D1 cells, but negatively correlates with cell migration and invasion ability. Besides, the miRNA-ribonucleoprotein complex AGO2/FXR1 was essential in the mechanism by which miR-320a regulated LRWD1 mRNA expression, and conclusively affects the ability of miR-320a to regulate LRWD1 expression in the absence of AGO2 or FXR1. Furthermore, miR-320a and LRWD1 were also responsive to oxidative stress, and that NRF2 was also regulated by the presence of miR-320a in response to ROS stimulation.
Conclusions: This is the first study showing miR-320a in upregulating testicular cancer-specific regulator LRWD1 and the importance of AGO2/FXR1 complex involved in miR-320a mediated upregulation of LRWD1 in testicular progression. |
| 關鍵字： | Testicular cancer; LRWD1; miR-320a; AGO2; FXR1 |
| 著作名稱： | 張章堂、莊一全、林宏明、黃珮珊、杜平惪、鄧燕妮∗，運用螢光細胞於環境污染物質的檢測，臺南大學環境與生態學報，第五卷，第一期，頁29-40。 |
| 年度： | 2012 |
| 類別： |
期刊論文
臺南大學環境與生態學報
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| 摘要： | 摘要
本研究運用具有綠色螢光蛋白 (Green Fluorescent Protein, GFP)的小鼠胚胎細胞（mouse embryonic feeder cells ，MEF）暴露在環境污染物檢測中，此螢光蛋白是一種高敏感度的螢光蛋白，實驗結果發現，在僅有50個細胞的情況下，即可偵測得到螢光。相對於一般實驗常用的MTT試驗，則需要1000個細胞以上，方能偵測得到細胞的存在。也就是說，GFP螢光細胞具有極高的敏感度及簡易偵測的優勢，在極低的細胞量就可以被偵測得到，有極高的潛力與優勢適合運用於環境毒物檢測中。以不同毒性物質H2O2、Benzo[a]pyrene（BaP)及來自環境中的柴油廢棄收集液等進行螢光試驗，細胞螢光強度與H2O2及Benzo[a]pyrene（BaP)的處理濃度呈負相關，顯示當毒性物質對於細胞毒殺作用越激烈時，細胞的螢光發散能力將受到影響。未來將可運用於其他領域的生物毒性檢測，包括：化妝品、中草藥、食品等，因此具有極高的外溢效應。此螢光細胞檢測技術平台的建立，不僅可以運用於環境污染物，利用螢光細胞之螢光發散的高敏感性，開發以螢光細胞作為檢測對象的快速檢測平台，藉由此檢測平台的建立，期望未來環境污染物的生物毒性檢測，能達到高敏感度及快速檢測的方向發展。
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| 關鍵字： | 小鼠胚胎細胞、綠色螢光細胞、生物毒性檢測 |
| 著作名稱： | Tumor Suppressor Protein P53 Regulates LRWD1 Expression in Testicular Cells |
| 年度： | 2023 |
| 類別： |
會議論文
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| 摘要： | The p53 protein, a tumor suppressor, is believed to impede cancer development by engaging in various signaling pathways. Our research findings indicate that LRWD1 possesses numerous p53 binding sites in its proximal promoter region. When p53 was overexpressed, we observed repression of LRWD1 promoter activity. Conversely, knockdown of p53 resulted in increased LRWD1 promoter activity and enhanced expression of LRWD1 protein. The binding of p53 to the -1230+93 promoter downregulated the expression of LRWD1. These findings strongly suggest that p53 binds to the LRWD1 promoter and hampers its expression.
Furthermore, in some patients with asthenozoospermia, we identified hypermethylation at the -208 position within the LRWD1 promoter, coinciding with a p53 binding site. The -208 (C>A) mutation significantly heightened the activity of the LRWD1 promoter, indicating that mutations at this position affect the p53 binding ability to the LRWD1 promoter. In summary, hypermethylation of the LRWD1 promoter and p53 binding contribute to the transcriptional suppression of LRWD1, leading to decreased LRWD1 expression. The interplay between DNA methylation, p53, and LRWD1 significantly regulates LRWD1 transcription. Identifying an association between p53-binding site mutations and the regulation of LRWD1 expression could enhance our understanding of male infertility and potentially find applications in reproductive medicine. |
| 關鍵字： | LRWD1, methylation, p53 |
| 著作名稱： | LRWD1 expression is regulated by MiR-320/AGO2 pathway in human testicular cells |
| 年度： | 2023 |
| 類別： |
會議論文
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| 摘要： | LRWD1 (Leucine-Rich repeats and WD repeat domain containing 1) exhibits high expression levels in the testes and localizes to the centrosome of sperm, where it plays a crucial role in microtubule growth. Through bioinformatics predictions using miRnada and miRnaMap software, binding sites for miR-320 were identified in the LRWD1 3 untranslated region (3UTR), suggesting potential regulatory interactions in reproductive tissues with elevated miR-320 expression.
To investigate the functional relationship between miR-320 and LRWD1, we constructed a fusion of the LRWD1 3UTR with the pMIR plasmid and performed a Dual-Luciferase Reporter Assay, which confirmed that miR-320 increased LRWD1 expression. Transfection of miR-320 mimic into NT2D1 cells further supported the upregulation of LRWD1 expression. Additionally, RNA-Binding Protein Immunoprecipitation (RIP) analysis validated the binding of miR-320a within the Ago2 protein. Overexpression of miR-320a mimic, AGO2, and FXR1 significantly enhanced the activity of the LRWD1 3UTR. Subsequent investigations demonstrated that LRWD1 and miR-320a directly suppressed cell invasion ability. In summary, LRWD1 safeguards DNA stability and mitigates DNA damage, with cellular responses to such damage primarily coordinated through the ATR-Chk1 pathways. Both p53 and miR-320a exert significant regulatory roles in modulating LRWD1 expression under oxidative stress. These findings provide valuable insights into the functional mechanisms and interactions between miR-320 and LRWD1 in post-transcriptional regulation, potentially facilitating the diagnosis and treatment of spermatogenic defects and male infertility conditions. |
| 關鍵字： | LRWD1, miR-320a, AGO2, FXR1 |
| 著作名稱： | Yen-Ni Teng, Yao-Chieh Hou, Hsing-Yi Chen, Chi-Ping Tai, Deng-Yu Tseng, Jie-Yu Tseng, Ming-Syuan Wu (2017, May). The role of LRWD1 in the embryonic development of zebrafish. 2017 European Society of Human Genetics, Copenhagen, Denmark. MOST 105-2314-B-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng, Ming-Syuan Wu, Hany A. Omar, Chia-Hui Su, Jie-Yun Tseng, Hsing-Yi Chen, Mei-Tsz Su, Po-Hsiu Wu (2017, May). MiR-320 regulated LRWD1 expression in human testicular cell. 2017 European Society of Human Genetics, Copenhagen, Denmark. MOST 105-2314-B-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng, Jie-Yun Tseng, Pao-Lin Kuo, Chia-Yun Wu, Min-Syuan Wu, Hsing-Yi Chen, Mei-Tsz Su, Yao-Chieh Hou, Chao-Chin Hsu (2017, May). Transcriptional regulation of Septin12. 2017 European Society of Human Genetics, Copenhagen, Denmark. MOST 106-2922-I-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng, Hsing-Yi Chen, Hany A. Omar, Yung-Ming Lin, Jie-Yun Tseng, Ming-Syuan Wu, Hsien-An Pan (2017, May). Expression of Human Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) by methylation. 2017 European Society of Human Genetics, Copenhagen, Denmark. MOST 105-2314-B-024-001. 本人為第一作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng, Hsing-Yi Chen, Hany A. Omar, Yung-Ming Lin, Jie-Yun Tseng, Ming-Syuan Wu, Hsien-An Pan (2017, May). Expression of Human Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) by methylation. 2017 European Society of Human Genetics, Copenhagen, Denmark. MOST 105-2314-B-024-001. 本人為第一作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng, Chia-Hui Su, Ming-Syuan Wu, Jie-Yun Tseng, Hsing-Yi Chen (2017, Jul). Transcriptional regulation of Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) by microRNA under Reactive Oxygen Species (ROS). 2017 15th Asia-Pacific Biotechnology Congress, Melbourne, Australia. MOST 105-2314-B-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2017 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Yen-Ni Teng*, Yin-Mei Su ,Xiu-Ying Liu, Yung-Chieh Tsai, Pao-Lin Kuo, Hsien-An Pan (2015, Apr). Transcriptional regulation and Expression of Human Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1). IFFS (International Federation of Fertility Societies) 2015 & 60th JSRM (Japan Society of Reproductive Medicine) Meeting 26–29 April 2015 Yokohama, Japan. 本人為第一作者、通訊作者 |
| 年度： | 2015 |
| 類別： |
會議論文
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| 摘要： | Study Question: Leucine-rich repeats and WD repeat domain containing protein 1 (LRWD1) is richly expressed in the testes, yet its roles and transcriptional regulation in human male germ cell development remain unclear. The objective of this study is to investigate the expression and function of LRWD1 in human sperms and the transcriptional regulation in human sperms.
Materials, Setting, Methods: We studied the expression of LRWD1 in sperm s of fertile males and infertile patients by RT-PCR and immunostaining. In addition, we further compared the expression of LRWD1 in the mature spermatozoa from normal controls and from patients with morphologically defective sperms. To assess whether the DNA methylation play an important role in transcriptional regulation of LRWD1, we analyzed the transcriptional activity and revealed the CpG islands of immediately upstream of LRWD1.
Main Results: In mature spermatozoa, we detected LRWD1 in the centrosome close to the connection region between the head and neck. The fraction of sperm with reduced level of LRWD1 was significantly increased in the semen samples of patients with asthenozoospermia, teratozoospermia, and asthenoteratozoospermia. Sperms without the LRWD1 signal were increased in the neck-defective and tail-defective groups. In addition, LRWD1 was highly expressed in the testes, and associated with the sperm morphology and motility. In the quantitative methylation-specific PCR (qMSP) showed the DNA hypermethylation of LRWD1 promoter was negatively correlated to sperm motility. Altogether, LRWD1expression is mediated by methylation in human sperms. |
| 關鍵字： | |
| 著作名稱： | Teng YN*, Wee SK, Su YM, Pan HA, Kuo PL, Hung JH, Tsao CC, Tsai YC, Fu TF, Hsu PC (2014, Apr). Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) is associated with Reactive oxygen species (ROS) response in testicular cells. The 5th Congress of the Asia Pacific Regional Initiative on Reproduction, Brisbane, Australia. MOST 102-2314-B-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2014 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Teng YN*, Su YM, Wee SK, Hung JH, Tsao CC, Tsai YC, Pan HA, Lin YM, Hsu PC, Chuang PJ, Chen SW (2014, Apr). Hypermethylation of the Leucine-Rich Repeats and WD Repeat Domain Containing 1 (LRWD1) Promoter in sperm. The 5th Congress of the Asia Pacific Regional Initiative on Reproduction, Brisbane, Australia. MOST 102-2314-B-024-001. 本人為第一作者、通訊作者. |
| 年度： | 2014 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Effects of 2,3′,4,4′,5-pentachlorobiphenyl (PCB 118) in SD rats testicular toxicity in F3 offspring. |
| 年度： | 2012 |
| 類別： |
會議論文
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| 摘要： | |
| 關鍵字： | |
| 著作名稱： | Chia-Hui Su, Ming-Syuan Wu, Jie-Yun Tseng, Yen-Ni Teng (2016, Aug). Reactive Oxygen Species (ROS) and miR-320a regulated LRWD1 expression. 台灣生殖醫學會年會. 本人為通訊作者. |
| 年度： | 2016 |
| 類別： |
會議論文
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| 摘要： | Study Question: LRWD1 (Leucine-Rich repeats and WD repeat domain containing 1) was highly expressed in the testes, and associated with the sperm morphology and motility. ROS are free radicals that are involved in numerous sperm physiological processes such as capacitation, hyper activation, and sperm-oocyte fusion. The objective of this study was to investigate the association between the reactive oxygen species and LRWD1 expression in testicular cell of human.
Study Design, Size, Duration: To assess whether the ROS play important role in LRWD1 of testicular cell human, we used hydrogen peroxide (H2O2) or sodium nitroprusside dehydrate (SNP) treatment in testicular cell, and investigated the role of miRNA for LRWD1 expression.
Materials, Setting, Methods:
Hydrogen peroxide (H2O2) or Sodium nitroprusside dehydrate (SNP) treatment to testicular cell to NT2D1 cell ,and then assay LRWD1 expression by western blot and real-time PCR. miR-320a have potential bind site on the 3’UTR of LRWD1 transcript by the microRNA analyzing tools, miRanda, TargetScan,Microcosm, StarBase and RNAhybrid. In the study, we reported the effects of LRWD1 on reactive oxygen species and miR-320a. We made LRWD1-3’UTR fused to a luciferase reporter vector to construct pMIR-LRWD1-3’UTR.
Main Results:
The expression of miR-320a and LRWD1 were increased by hydrogen peroxide (H2O2) or sodium nitroprusside dehydrate (SNP) treatment in TaqMan real time-PCR assay.
Conclusion: With this study, the post-transcriptional regulation of miR-320a for LRWD1 help to understand the function and roles of the miRNA in post-transcriptional regulation of LRWD1 and may provide a rationale for further diagnosis and treatment of spermatogenic defect and male infertility diseases.
Acknowledgment
This study was supported by grants from National Science Council (NSC 102-2314-B-024 -001) and Ministry of Science and Technology (MOST 103-2314-B-024 -002, MOST 104-2314-B-024 -002 -MY2) of Taiwan. |
| 關鍵字： | |
| 著作名稱： | Human LRWD1 Is Associated with Centrosomes and Microtubules Organization |
| 年度： | 2013 |
| 類別： |
會議論文
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| 摘要： | LRWD1 (Leucine-Rich repeats and WD repeat domain containing 1) is highly expressed in the testes, but the roles in male germ cell development and spermatogenesis remain unclear. The objective of this study is to investigate the expression and function of LRWD1 in human sperms. We studied the expression of LRWD1 in fertile males and infertile patients by RT-PCR and immunostaining. LRWD1 was detected in the testes of all human subjects except Sertoli-cell-only-syndrome (SCOS) patients. In spermatozoa, LRWD1 was found to colocalized with centrin and γ-tubulin in the centrosome region. We also applied the immunofluorescence assay (IFA) to study the expression of LRWD1 in ejaculated spermatozoa of various patients with sperm abnormality. In the neck- and tail-defective groups, we found that significant reduction of sperms LRWD1 immunostaining signals.
Sperm tails is the structure extending from centrosome. LRWD1 localizes at the centrosome in human spermatozoa and correlates with multiple sperm morphological defects according to the decrease of LRWD1. We hypothesized that human LRWD1 is a centrosomal protein and plays an important role for the microtubule organization in the cell cytoskeleton and sperm tails formation. Knockdown of LRWD1 in HeLa cells by siRNA demonstrated that LRWD1 participates in microtubule organization and function. In IFA experiment, the LRWD1 colocalize with γ-tubulin in the centrosome region. Rescue after vinblastine treatment, LRWD1 have more signal on the centrosome. We suggest that LRWD1 may have important role in cell cytoskeleton formation. |
| 關鍵字： | |
| 著作名稱： | 男性不孕症患者LRWD1的 p53RE分析及相關研究 |
| 年度： | 2023 |
| 類別： |
研究計畫成果報告
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| 摘要： | Leucine-rich repeats and WD repeat domain containing 1 (LRWD1)於睪丸組織中高度表現，LRWD1在氧化壓力下可以維持DNA的穩定與降低DNA損傷，並且LRWD1啟動子上含有多個p53結合位點(p53 RE)，因此本研究將p53過表達後發現p53可抑制LRWD1 啟動子的活性以及LRWD1蛋白的表現，而阻斷（knockdown） p53增加LRWD1 啟動子的活性以及LRWD1蛋白的表現，推測當p53過表達時LRWD1轉錄受到p53的抑制，而降低LRWD1表現；而當阻斷p53表現時，p53無法有效率抑制LRWD1啟動子進而增加LRWD1表現。以不同LRWD1啟動子長度(-1230+93、-475+93)觀察p53對LRWD1啟動子活性影響，發現LRWD1啟動子區域從-1230至+93皆明顯受到p53轉錄抑制作用降低LRWD1啟動子活性，p53可結合於LRWD1啟動子區域並抑制其表現。
活動力較低精子LRWD1啟動子-208位點(-208位點位於p53結合區域)， 在啟動子轉錄活性分析顯示LRWD1啟動子上p53結合區域-208位點突變大幅增加LRWD1啟動子的活性，推測為-208(C>A)突變影響p53結合於LRWD1啟動子的能力，進而改變p53對LRWD1的調控。在以p53結合位突變的R248W 選植體過表現在 NT2D1 (p53 +/+)細胞中，結果顯示LRWD1的表現顯著提高。綜合以上研究結果p53結合於LRWD1啟動子區域可抑制LRWD1轉錄，進而抑制LRWD1的表現，p53對於LRWD1的轉錄調控與表現扮演重要角色。 |
| 關鍵字： | LRWD1基因， p53反應位置, 啟動子 |
| 著作名稱： | p53和NRF2在LRWD1的轉錄調節和抗氧化活性的作用 |
| 年度： | 2021 |
| 類別： |
研究計畫成果報告
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| 摘要： | LRWD1大量表現於睪丸組織且嚴重造精功能障礙患者則表現顯著偏低
。當阻斷LRWD1表現時，細胞停滯於G1期且DNA損傷增加。LRWD1的基
因表現隨著細胞中p53促進劑或抑制劑的處理而增加或減少，且生物
資訊軟體PROMO3.0軟體分析LRWD1啟動子區域，結果發現在LRWD1 的
-165/-159、-66/-60、-39/-28位置有p53的結合位點，以Chromatin
Immunoprecipitation assay (ChIP)與DNA affinity purification
assay (DAPA)實驗證實p53轉錄因子會與LRWD1啟動子有穩定的結合
。以p53 抑制劑 (Pifithrin-α)處理生殖細胞抑制p53活化，發現
LRWD1轉錄活性在處理時有明顯下降的趨勢，因此當p53被抑制時
，會造成LRWD1啟動子活性下降，而無法進行轉錄的調控。在對於細
胞週期影響的研究顯示，無論在有無p53刺激下，只要LRWD1被剔除
的情況下，細胞均停滯在G1期。在對於DNA損傷的影響方面，當
LRWD1被剔除的細胞中，無論過度表現或抑制p53生成，都會造成
DNA損傷，影響LRWD1表現，推測p53調控LRWD1的表現，降低DNA損傷
對於細胞後續的影響，以維護DNA的完整及正確性。LRWD1 的表達受
mir-320a 和 NRF2 的調控。當 LRWD1 或 NRF2 分別被阻斷時，細
胞侵襲顯著增加或減少。在氧化壓力下mir-320a、NRF2和p53對
LRWD1表達和細胞侵襲的調控，則有待後續深入研究。總結p53是影
響LRWD1基因表現的重要因素，藉由ChIP及啟動子活性分析確認
p53轉錄因子對於LRWD1轉錄活性的影響，確認LRWD1受p53轉錄因子
調控及在細胞抗氧化壓力的角色及功能。藉由本研究的完成，將有
助於了解p53對於LRWD1表現的調控及在細胞對抗氧化壓力的機制
，除了在生殖方面，也可以作為研究氧化壓力相關疾病的參考。 |
| 關鍵字： | ： LRWD1基因、p53基因、NRF2基因、活性氧自由基 |
| 著作名稱： | 探討LRWD1在睾丸維護DNA完整的角色 |
| 年度： | 2019 |
| 類別： |
研究計畫成果報告
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| 摘要： | LRWD1大量表現於睪丸組織且嚴重造精功能障礙患者則顯著偏低，當
LRWD1受阻斷時，細胞停滯於G1期，此細胞受到DNA損傷的壓迫時
，DNA損傷程度明顯高於LRWD1表現正常的細胞。以生物資訊軟體預
測顯示鄰近LRWD1啟動子區域約有10個p53轉錄因子的結合位置，推
測LRWD1可能參與DNA損傷及修補作用，以確保睪丸細胞DNA的正確及
完整性。
本研究探討LRWD1在睾丸維護DNA完整的角色，以shRNA阻斷細胞中
LRWD1的表現或以pEGFP-hLRWD1轉染到細胞中大量表現LRWD1，證明
LRWD1在睾丸細胞維護DNA完整的角色。另外， p53對於LRWD1在細胞
受到氧化自由基壓力時，調控LRWD1表現以避免細胞DNA受損及細胞
損傷。在有氧化自由基的刺激下，LRWD1具有避免DNA受損及保護細
胞的作用，減少細胞死亡，本研究成果釐清LRWD1在細胞維護DNA完
整的角色，期待未來能作為精液品質改善及臨床運用的參考。 |
| 關鍵字： | 睪丸細胞，DNA完整性，活性氧 |
| 著作名稱： | 卵子受精及胚胎發育過程中miR-320及LRWD1在氧化壓力反應的功能與角色 |
| 年度： | 2018 |
| 類別： |
研究計畫成果報告
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| 摘要： | 卵巢功能維持與卵子生成是複雜且需要許多基因協助才能完成的過
程，卵巢細胞內的氧化自由基的濃度與氧化壓力是影響的卵巢功能
的重要因素。MiR-320可調控抗氧化基因的表現，以減少氧化壓力對
於卵巢細胞的傷害。本研究顯示在人類卵巢細胞株OVCAR-3及豬的卵
丘細胞中，MiR-320調控LRWD1的表現，減少氧化壓力對於睪丸細胞
的傷害。在氧化壓力的環境下，MiR-320和LRWD1的表現增加，藉由
將LRWD1 3’UTR構築在pMIR轉殖質體並藉由冷光報導基因的分析
，了解在氧化壓力下MiR-320對於LRWD1的表現調控；藉由MiR-320
mimic/inhibitor及LRWD1阻斷實驗，顯示MiR-320具有調控LRWD1表
現，以調控細胞在H2O2或SNP處理等氧化壓力對於細胞的影響。在卵
子生成及對抗氧化壓力上，具有極大的意義。藉由本研究的完成
，了解能在抗氧化過程中MiR-320和LRWD1的角色，對於MiR-320和
LRWD1在卵巢癌變及卵巢中氧化壓力等造成的女性卵巢疾病中的角色
，應該具有極大的參考及運用價值。 |
| 關鍵字： | LRWD1、miR-320、氧化壓力 |
| 著作名稱： | SEPT12複合體在造精功能所扮演的角色-Septin12的轉錄調控 |
| 年度： | 2017 |
| 類別： |
研究計畫成果報告
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| 摘要： | 在造精過程中SEPTIN12基因的轉錄調控機制目前仍不清楚。為了要
釐清SEPTIN12的轉錄調控機制，首先以PROMO3.0軟體分析
SEPTIN12啟動子區域，結果發現在SEPTIN12有雌激素受體(estrogen
receptor，ER)和雄性素受體(androgen receptor，AR)的結合位。
以染色質免疫共沉澱實驗證實ER及AR會結合在SEPTIN12啟動子區域
。將SEPTIN12啟動子上的ER或AR結合位點進行特定位置突變，使
AR或ER無法結合在SEPTIN12上，在轉錄活性分析顯示SEPTIN12啟動
子活性明顯降低。以雌激素(estrogen, E2)或雄性素(androgen,
DHT)處理細胞以誘導ER與AR活化，發現SEPTIN12轉錄活性在處理後
有明顯上升的趨勢。將AR與ER構築質體轉染到細胞中，使受體過度
表現於細胞後給予DHT或E2刺激，SEPTIN12轉錄活性有明顯上升，但
SEPTIN12啟動子上ER或AR結合位點進行突變之組別再轉染AR與ER使
受體過度表現後給予DHT或E2時卻無法提升SEPTIN12轉錄活性。此外
本研究以15種雌性素及雄性素結構相似的環境荷爾蒙(endocrine
disrupting chemicals, EDC)處理細胞以誘導ER與AR活化，發現
SEPTIN12轉錄活性在處理環境荷爾蒙時發現
EDC1,2,3,4,8,9,10,13,14有明顯上升的趨勢，將此15種環境賀爾蒙
與E2或DHT共同處理，發現EDC 2與E2共同處理明顯降低SEPTIN12轉
錄活性，而EDC 1,3,9與DHT共同處理則為最顯著影響SEPTIN12轉錄
活性，並利用AR拮抗劑或ER拮抗劑與15個EDC共同處理，發現15個
EDC與ER拮抗劑共同處理只有EDC 1跟EDC 3沒有受到拮抗劑的影響。
在男性不育中發現有單核苷酸多型性(single nucleotide
polymorphisms, SNP)的點rs3747608及rs759992在SEPTIN12啟動子
區域，而在NCBI中rs3827527也位於SEPTIN12核心啟動子的AR結合位
置，以點進行特定位置突變方式將rs3747608 , rs759992及
rs3827527這些SNPs位點導入在SEPTIN12核心啟動子後轉染到細胞中
，顯示SEPTIN12轉錄活性有顯著下降。總結以上，雌激素或雄性素
會藉由AR與ER調控SEPTIN12轉錄活性及基因表現，而AR與ER也會受
到環境賀爾蒙所影響而影響SEPTIN12轉錄活性，部分的環境荷爾蒙
會與DHT或E2有交互作用而影響SEPTIN12的基因表現。單核苷酸多型
性在不育患者中沒有廣泛的存在，但利用啟動子活性分析發現這兩
個SNP點顯著降低SEPTIN12的轉錄活性。 |
| 關鍵字： | SEPTIN12 雌激素 雄性素 單核苷酸多型性 環境荷爾蒙 |
| 著作名稱： | 微小核醣核酸對於睪丸細胞的LRWD1基因表現與功能影響 |
| 年度： | 2017 |
| 類別： |
研究計畫成果報告
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| 摘要： | Micro RNA是後轉錄調節的重要機制，且決定基因在mRNA的表達或抑
制的重要角色，本實驗以miRanda等軟體預測LRWD1 3’UTR的micro
RNA預測，顯示在miR-320a與miR-450a在其序列上結合的機會較高
，再用miRnaMap等軟體搜尋現有的miR-320a和miR-450a等資料，發
現miR-320a及miR-450a在生殖相關的組織具有較高的表現。將LRWD1
3’UTR的序列構築在pMIR質體，在利用雙螢光素酶報告基因檢測系
統顯示miR-320a 顯著提高LRWD1的表現3’UTR，但在miR-450a則沒
有明顯差異。在將miR-320a 和miR-450a轉染到NTERA-2
cl.D1（NT2D1）之後利用蛋白質電泳和real time PCR 分析LRWD1蛋
白質及mRNA的表現量，結果顯示miR-320a能夠提高LRWD1在蛋白質及
mRNA的表現。另外在過氧化氫（hydrogen peroxide, H2O2）及
Sodium nitroprusside dehydrate（SNP）處理NT2D1後，利用
TaqMan real time-PCR分析，結果顯示miR-320a和LRWD1的表現顯著
增加。將miR-320a和miR-450a轉染到NT2D1細胞後，結果顯示當過量
表現miR-320a時會使NT2D1的生長速度增快。在LRWD1 3’UTR的單一
核苷酸多型性分析（SNPs）中，在基因資料庫的分析結果顯示35個
SNPs位於LRWD1 3’UTR中，其中有7個SNPs位於在是都並在 miR320a結合位置上，發現包括在我們的研究樣品中有17個SNPs，但是
都並未表現在 miR-320a結合位置上，且經過統計分析顯示與不孕症
患者沒有直接的統計連結意義，顯示LRWD1 3’UTR的單一核苷酸多
型性分析（SNPs）不是台灣男性生殖障礙的主因素。總結本研究成
果，雖然在台灣病人的LRWD1 3’UTR上的單一核苷酸多型性分析結
果，沒有顯示與造精功能障礙患者的直接關係，但在細胞學研究中
，miR-320a能結合在LRWD1 3’UTR上，並且影響LRWD1的基因表現活
性，過氧化氫（hydrogen peroxide, H2O2）及Sodium
nitroprusside dehydrate（SNP）等造成細胞自由基的處理確實會
使影響miR-320a表現進而影響LRWD1的表現，顯示氧化自由基確實能
促進LRWD1表現進而影響細胞LRWD1的表現及功能。未來希望將本研
究成果運用在生殖及基因表現模式的探討中，甚至運用在臨床的檢
測中，對人類生殖的研究有所助益。 |
| 關鍵字： | LRWD1、miR-320a、miR-450a、男性不孕症 |
| 著作名稱： | 活性氧對於睪丸細胞的LRWD1基因表現與功能影響 |
| 年度： | 2015 |
| 類別： |
研究計畫成果報告
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| 摘要： | LRWD (Leucine-Rich repeats and WD repeat domain containing
1) 基因主要表現於睪丸組織中，尤其是精蟲的中心體特別明顯，推
測此些表現特點與轉錄調控有直接關係。LRWD1的轉錄啟動子位置9至-5 bp具有Nrf2轉錄因子結合位。Nrf2可結合於ARE，並誘導解毒
酵素II與抗氧化酵素等的表現以對抗環境中ROS。在Chromatin
Immunoprecipitation（ChIP）和electrophoretic mobility shift
assays （EMSA）實驗中證實了Nrf2會與LRWD1啟動子上游區域結合
。在將LRWD1的Nrf2結合位點突變後，由Dual luciferase reporter
system分析啟動子之活性確實明顯下降低許多，可能由於Nrf2轉錄
因子無法與啟動子結合，因而明顯降低LRWD1啟動子活性，Nrf2是
LRWD1基因啟動子的重要轉錄因子之一。過氧化氫（Hydrogen
peroxide）及Sodium pentacyanonitrosylferrate (III)
dehydrate（釋放一氧化氮）藥物處理HeLa細胞以誘發Nrf2活化，發
現LRWD1蛋白與mRNA表現量也有明顯的上升，即當環境中的ROS濃度
提升，Nrf2及LRWD1蛋白表現量也因此提升。總結以上，我們認為
Nrf2為調控LRWD1啟動子的重要轉錄因子。
進一步分析LRWD1核心啟動子區域(-198~+1)附近區域的DNA甲基化修
飾情況，在生物資訊軟體EMBOSS CpG Plot分析顯示LRWD1啟動子253到+5之間是CpG islands區域，且此區域包含NF-κB與NRF2轉錄
因子結合位。將LRWD1基因-400到+93序列接載到帶有螢光蟲報導基
因之pCpGL-basic vector上，形成pCpGL-hLRWD1 promoter -
400/+93 vector。在帶有LRWD1啟動子之載體以甲基化酵素SssI進行
甲基化後，轉殖入真核NT2D1或是HeLa細胞並透過Luciferase
activity assay的活性分析顯示啟動子區域被甲基化後，細胞的啟
動子活性及基因表現下降。透過Quantitative methylationspecific PCR(qMSP) 分析LRWD1啟動子NRF2轉錄因子結合位的甲基
化與精液品質的關係，顯示NRF2轉錄因子結合區域的甲基化與精子
活動力呈現負相關。綜合以上結果，LRWD1受活性氧的刺激而加強表
現， Nrf2是LRWD1重要的轉錄因子，LRWD1轉錄調控位置的變液或甲
基化會造成LRWD1轉錄活性下降，基因的表現降低。LRWD1是精蟲結
構的重要蛋白，以上LRWD1受活性氧影響的重要研究成果，對於不孕
症的致因探討及未來的臨床諮商與預防，將有重要的參考價值。 |
| 關鍵字： | LRWD1基因、活性氧（reactive oxygen species，ROS）甲基化 |