TGF-ß1 and TGF-ß3, but not TGF-ß2, are upregulated in the ovaries of ovarian hyperstimulation syndrome†.

Ovarian hyperstimulation syndrome (OHSS) is a life-threatening and potentially fatal complication during in vitro fertilization treatment. The levels of transforming growth factor-ß1 (TGF-ß1) are upregulated in human follicular fluid and granulosa-lutein cells (hGL) of OHSS patients and could contribute to the development of OHSS by downregulating steroidogenic acute regulatory protein (StAR) expression. However, whether the same is true for the other two members of the TGF-ß family, TGF-ß2 and -ß3, remains unknown. We showed that all three TGF-ß isoforms were expressed in human follicular fluid. In comparison, TGF-ß1 was expressed at the highest level, followed by TGF-ß2 and TGF-ß3. Compared to non-OHSS patients, follicular fluid levels of TGF-ß1 and TGF-ß3 were significantly upregulated in OHSS patients. The same results were observed in mRNA levels of TGF-ß isoforms in hGL cells and ovaries of OHSS rats. In addition, StAR mRNA levels were upregulated in hGL cells of OHSS patients and the ovaries of OHSS rats. Treatment cells with TGF-ß isoforms downregulated the StAR expression with a comparable effect. Moreover, activations of SMAD3 signaling were required for TGF-ß isoforms-induced downregulation of StAR expression. This study indicates that follicular fluid TGF-ß1 and TGF-ß3 levels could be used as biomarkers and therapeutic targets for the OHSS.

NPFF stimulates human ovarian cancer cell invasion by upregulating MMP-9 via ERK1/2 signaling.

Neuropeptide FF (NPFF) belongs to the RFamide peptide family. NPFF regulates a variety of physiological functions by binding to a G protein-coupled receptor (GPCR), NPFFR2. Epithelial ovarian cancer (EOC) is a leading cause of death among gynecological malignancies. The pathogenesis of EOC can be regulated by many local factors, including neuropeptides, through an autocrine/paracrine manner. However, to date, the expression and/or function of NPFF/NPFFR2 in EOC is undetermined. In this study, we show that the upregulation of NPFFR2 mRNA was associated with poor overall survival in EOC. The TaqMan probe-based RT-qPCR showed that NPFF and NPFFR2 were expressed in three human EOC cells, CaOV3, OVCAR3, and SKOV3. In comparison, NPFF and NPFFR2 expression levels were higher in SKOV3 cells than in CaOV3 or OVCAR3 cells. Treatment of SKOV3 cells with NPFF did not affect cell viability and proliferation but stimulated cell invasion. NPFF treatment upregulates matrix metalloproteinase-9 (MMP-9) expression. Using the siRNA-mediated knockdown approach, we showed that the stimulatory effect of NPFF on MMP-9 expression was mediated by the NPFFR2. Our results also showed that ERK1/2 signaling was activated in SKOV3 cells in response to the NPFF treatment. In addition, blocking the activation of ERK1/2 signaling abolished the NPFF-induced MMP-9 expression and cell invasion. This study provides evidence that NPFF stimulates EOC cell invasion by upregulating MMP-9 expression through the NPFFR2-mediated ERK1/2 signaling pathway.

TGF-ß1 upregulates secreted protein acidic and rich in cysteine expression in human granulosa-lutein cells: a potential mechanism for the pathogenesis of ovarian hyperstimulation syndrome.

BACKGROUND: Ovarian hyperstimulation syndrome (OHSS) is a serious complication during in vitro fertilization (IVF) treatment. The upregulation of ovarian transforming growth factor-beta 1 (TGF-ß1) is involved in the development of OHSS. The secreted protein acidic and rich in cysteine (SPARC) is a secreted multifunctional matricellular glycoprotein. Although the regulatory effects of TGF-ß1 on SPARC expression have been reported, whether TGF-ß1 regulates SPARC expression in the human ovary remains unknown. In addition, the role of SPARC in the pathogenesis of OHSS is unclear. METHODS: A steroidogenic human ovarian granulosa-like tumor cell line, KGN, and primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing IVF treatment were used as experimental models. OHSS was induced in rats, and ovaries were collected. Follicular fluid samples were collected from 39 OHSS and 35 non-OHSS patients during oocyte retrieval. The underlying molecular mechanisms mediating the effect of TGF-ß1 on SPARC expression were explored by a series of in vitro experiments. RESULTS: TGF-ß1 upregulated SPARC expression in both KGN and hGL cells. The stimulatory effect of TGF-ß1 on SPARC expression was mediated by SMAD3 but not SMAD2. The transcription factors, Snail and Slug, were induced in response to the TGF-ß1 treatment. However, only Slug was required for the TGF-ß1-induced SPARC expression. Conversely, we found that the knockdown of SPARC decreased Slug expression. Our results also revealed that SPARC was upregulated in the OHSS rat ovaries and in the follicular fluid of OHSS patients. Knockdown of SPARC attenuated the TGF-ß1-stimulated expression of vascular endothelial growth factor (VEGF) and aromatase, two markers of OHSS. Moreover, the knockdown of SPARC reduced TGF-ß1 signaling by downregulating SMAD4 expression. CONCLUSIONS: By illustrating the potential physiological and pathological roles of TGF-ß1 in the regulation of SPARC in hGL cells, our results may serve to improve current strategies used to treat clinical infertility and OHSS. Video Abstract.

Biomarkers identification in follicular fluid of women with OHSS by using UPLC-MS method.

To figure out the differentially changed metabolites and disturbed pathways in follicular fluid (FF) of patients with OHSS in comparison to the control group undergoing in vitro fertilization (IVF), we conducted this metabolomic analysis between two groups, the OHSS group included 30 patients treated with oocyte retrieval and developed OHSS in the next 7-14 days, while another 30 patients without OHSS tendency were selected as the control group. The FF samples were obtained during the process of oocyte retrieval. FF samples were analyzed using ultra-high liquid chromatography-tandem mass spectrometry (UPLC-MS). The results identified a total of 59 differentially changed metabolites, including 33 decreased metabolites (P < 0.01) and 26 increased metabolites (P < 0.01) in FF of OHSS compared with the control group. 12 metabolites could be the most valuable biomarkers for OHSS based on ROC results. Our correlation analyses showed that deoxyinosine levels were found positively correlated with serum estradiol (E2) levels in OHSS patients, while L-isoleucine, pyruvic acid, maleamate, and arachidonic acid were found to be positively correlated with the number of retrieved oocytes. Furthermore, 4-hydroxyphenylacetaldehyde, deoxycorticosterone, creatinine, and creatine were found to be negatively associated with serum E2 levels, while 4-hydroxyphenylacetaldehyde, L-carnitine, isovaleric acid and L-2-hydroxyglutaric acid were negatively related with the number of oocytes retrieved in OHSS patients. Taken together, our study provides better identification of OHSS FF metabolic dynamics, suggesting the metabolic compounds can be used as valuable predictors or treatment targets of OHSS.

A meta-analysis of serum lipid profiles in premature ovarian insufficiency.

Premature ovarian insufficiency (POI), defined as loss of normal ovarian functions before the age of 40 years, occurs in at least 1% of all women. It affects the reproductive system and causes many health problems and psychological stress. Abnormal serum lipid profile leads to cardiovascular diseases, which are strongly associated with high mortality in patients with POI. To date, several studies have examined the levels of different serum lipids in patients with POI. The results, however, are either inconclusive or inconsistent. Therefore, the aim of this meta-analysis was to measure whether serum levels of total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) and triglyceride are varied in patients with POI. Ten studies in total were included in this meta-analysis involving 1009 individuals: 458 patients with POI and 551 controls. Our analysis results showed that serum total cholesterol (P < 0.00001), LDL-C (P < 0.0001), and triglyceride (P = 0.01) levels were significantly higher in patients with POI compared with healthy controls. Serum HDL-C levels, however, did not vary significantly between controls and patients with POI. These results suggest that elevations in unfavourable lipids may contribute to the high risk of cardiovascular diseases that are observed in patients with POI.

Growth differentiation factor-11 downregulates steroidogenic acute regulatory protein expression through ALK5-mediated SMAD3 signaling pathway in human granulosa-lutein cells.

BACKGROUND: Growth differentiation factor-11 (GDF-11) belongs to the transforming growth factor-ß (TGF-ß) superfamily. To date, the expression of GDF-11 in the ovary and its role in regulating ovarian function are completely unknown. Ovarian granulosa cell-mediated steroidogenesis plays a pivotal role in maintaining normal female reproductive function. GDF-11 and GDF-8 share high sequence similarity and exhibit many similar features and functions. Steroidogenic acute regulatory protein (StAR) regulates the rate-limiting step in steroidogenesis and its expression can be downregulated by GDF-8. Polycystic ovary syndrome (PCOS) is the most common cause of female infertility. The expression levels of GDF-8 are upregulated in the human follicular fluid and granulosa-lutein (hGL) cells of PCOS patients. However, whether similar results can be observed for the GDF-11 needs to be determined. METHODS: The effect of GDF-11 on StAR expression and the underlying molecular mechanisms were explored by a series of in vitro experiments in a primary culture of hGL cells obtained from patients undergoing in vitro fertilization (IVF) treatment. Human follicular fluid samples were obtained from 36 non-PCOS patients and 36 PCOS patients. GDF-11 levels in follicular fluid were measured by ELISA. RESULTS: GDF-11 downregulates StAR expression, whereas the expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) are not affected by GDF-11 in hGL cells. Using pharmacological inhibitors and a siRNA-mediated approach, we reveal that ALK5 but not ALK4 mediates the suppressive effect of GDF-11 on StAR expression. Although GDF-11 activates both SMAD2 and SMAD3 signaling pathways, only SMAD3 is involved in the GDF-11-induced downregulation of StAR expression. In addition, we show that SMAD1/5/8, ERK1/2, and PI3K/AKT signaling pathways are not activated by GDF-11 in hGL cells. RT-qPCR and ELISA detect GDF-11 mRNA expression in hGL cells and GDF-11 protein expression in human follicular fluid, respectively. Interestingly, unlike GDF-8, the expression levels of GDF-11 are not varied in hGL cells and follicular fluid between non-PCOS and PCOS patients. CONCLUSIONS: This study increases the understanding of the biological function of GDF-11 and provides important insights into the regulation of ovarian steroidogenesis.

Epigallocatechin-3-gallate stimulates StAR expression and progesterone production in human granulosa cells through the 67-kDa laminin receptor-mediated CREB signaling pathway.

Epigallocatechin-3-gallate (EGCG) is the most abundant and biologically active catechins extracted from green tea. The health benefits of EGCG have been extendedly studied. Ovarian steroidogenesis plays a pivotal role in maintaining normal reproductive function. Granulosa cells in the ovary are essential for steroid hormone production. To date, the effect of EGCG on steroidogenesis in human granulosa cells remains unclear. In the present study, we examine the physiological concentrations of EGCG on steroidogenesis in a steroidogenic human granulosa-like tumor cell line, KGN. Our results demonstrate that treatment with EGCG upregulates steroidogenic acute regulatory protein (StAR) expression and increases progesterone (P4) production. EGCG does not affect the expression levels of other steroidogenesis-related enzymes, such as P450 side-chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, and aromatase. In addition, we identify the expression of 67-kDa laminin receptor (67LR) in KGN cells. Moreover, EGCG-induced StAR expression and P4 production require the 67LR-mediated activation of the PKA-CREB signaling pathway. These results provide a better understanding of the function of EGCG on ovarian steroidogenesis, which may lead to the development of alternative therapeutic approaches for reproductive disorders.

G protein-coupled estrogen receptor stimulates human trophoblast cell invasion via YAP-mediated ANGPTL4 expression.

Insufficient invasion of trophoblast cells into the uterine decidua is associated with preeclampsia (PE). G protein-coupled estrogen receptor (GPER) is a membrane estrogen receptor involved in non-genomic estrogen signaling. GPER is expressed in human trophoblast cells and downregulated GPER levels are noted in PE. However, to date, the role of GPER in trophoblast cells remains largely unknown. Here, we applied RNA sequencing (RNA-seq) to HTR-8/SVneo human trophoblast cells in response to G1, an agonist of GPER, and identified angiopoietin-like 4 (ANGPTL4) as a target gene of GPER. Treatment of trophoblast cells with G1 or 17ß-estradiol (E2) activated Yes-associated protein (YAP), the major downstream effector of the Hippo pathway, via GPER but in a mammalian STE20-like protein kinase 1 (MST1)-independent manner. Using pharmacological inhibitors as well as loss- and gain-of-function approaches, our results revealed that YAP activation was required for GPER-stimulated ANGPTL4 expression. Transwell invasion assays demonstrated that activation of GPER-induced ANGPTL4 promoted cell invasion. In addition, the expression levels of GPER, YAP, and ANGPTL4 were downregulated in the placenta of patients with PE. Our findings reveal a mechanism by which GPER exerts its stimulatory effect on human trophoblast cell invasion by upregulating YAP-mediated ANGPTL4 expression.

BMP-9 downregulates StAR expression and progesterone production by activating both SMAD1/5/8 and SMAD2/3 signaling pathways in human granulosa-lutein cells obtained from gonadotropins induced ovarian cycles.

Bone morphogenetic proteins (BMPs) are expressed in different cell types of the human ovarian follicle and play important roles in the regulation of ovarian function. BMP-9, also known as growth differentiation factor-2 (GDF-2), belongs to the transforming growth factor-beta (TGF-ß) superfamily. BMP-9 is mainly synthesized in the liver and secreted into the blood which allows it to regulate various physiological and pathological functions. To date, the expression of BMP-9 in the human ovary and its function in human granulosa cells remains unknown. In the present study, we detect the protein expression of BMP-9 in the human follicular fluid. Using the primary culture of human granulosa-lutein (hGL) cells obtained from patients undergoing in vitro fertilization as a cell model, we show that treatment with BMP-9 downregulates steroidogenic acute regulatory protein (StAR) expression and suppresses progesterone (P4) production. The expression levels of the P450 side-chain cleavage enzyme (P450scc) and 3ß-hydroxysteroid dehydrogenase (3ß-HSD) are not affected by BMP-9 treatment. Mechanistically, treatment of hGL cells with BMP-9 activates both SMAD1/5/8 and SMAD2/3 signaling pathways. Blocking the activations of SMAD1/5/8 and SMAD2/3 by pharmacological inhibitors or knockdown of SMAD4 attenuates the inhibitory effects of BMP-9 on StAR expression and P4 production. This study reveals a novel function of BMP-9 in the regulation of ovarian steroidogenesis.

High ovarian GDF-8 levels contribute to elevated estradiol production in ovarian hyperstimulation syndrome by stimulating aromatase expression.

Rationale: Growth differentiation factor-8 (GDF-8), also known as myostatin, belongs to the transforming growth factor-beta (TGF-ß) superfamily. GDF-8 is expressed in the ovary and regulates various ovarian functions. Ovarian hyperstimulation syndrome (OHSS) is one of the most serious disorders during in vitro fertilization treatment. Aromatase, encoded by the CYP19A1 gene, is the enzyme that catalyzes the final step in estradiol (E2) biosynthesis. It has been demonstrated that high serum E2 levels are associated with the development of OHSS. However, the effects of GDF-8 on aromatase expression and its roles in the pathogenesis of OHSS remain unclear. Methods: The effect of GDF-8 on aromatase expression and the underlying mechanisms were explored by a series of in vitro experiments in primary human granulosa-lutein (hGL) and KGN cells. Rat OHSS model and human follicular fluid samples were used to examine the roles of the GDF-8 system in the pathogenesis of OHSS. Results: We demonstrate that GDF-8 stimulates aromatase expression and E2 production in hGL and KGN cells. In addition, TGF-ß type I receptor ALK5-mediated SMAD2/3 signaling is required for GDF-8-induced aromatase expression and E2 production. Using a rat OHSS model, we show that the aromatase and GDF-8 levels are upregulated in the ovaries of OHSS rats. Blocking the function of ALK5 by the administration of its inhibitor, SB431542, alleviates OHSS symptoms and the upregulation of aromatase. Clinical results reveal that the protein levels of GDF-8 are upregulated in the follicular fluid of OHSS patients. Moreover, the expression of GDF-8 is increased in hGL cells of OHSS patients. Conclusions: This study helps to elucidate the mechanisms mediating the expression of aromatase in human granulosa cells, which may lead to the development of alternative therapeutic approaches for OHSS.

Ovarian Hyperstimulation Syndrome Is Associated with a High Secondary Sex Ratio in Fresh IVF Cycles with Cleavage-Stage Embryo Transfer: Results for a Cohort Study.

The sex ratio at birth is defined as the secondary sex ratio (SSR). Ovarian hyperstimulation syndrome (OHSS) is a serious and iatrogenic complication associated with controlled ovarian stimulation (COS) during assisted reproductive technology (ART) treatments. It has been hypothesized that the human SSR is partially controlled by parental hormone levels around the time of conception. Given the aberrant hormonal profiles observed in patients with OHSS, this retrospective study was designed to evaluate the impact of OHSS on the SSR. In this study, all included patients were divided into 3 groups: non-OHSS (n=2777), mild OHSS (n=644), and moderate OHSS (n=334). Our results showed that the overall SSR for the study population was 1.033. The SSR was significantly increased in patients with moderate OHSS (1.336) compared to non-OHSS patients (1.002) (p=0.048). Subgroup analyses showed that increases in the SSR in patients with moderate OHSS were observed in the IVF group (1.323 vs 1.052; p=0.043), but not in the ICSI groups (1.021 vs 0.866; p=0.732). In addition, the elevated serum estradiol (E2) and progesterone (P4) levels in OHSS patients were not associated with SSR. In this study, for the first time, we report that a high SSR is associated with OHSS in patients who received fresh IVF treatments. The increases in SSR in OHSS patients are not attributed to the high serum E2 and P4 levels. Our findings may make both ART clinicians and patients more aware of the influences of ART treatments on the SSR and allow clinicians to counsel patients more appropriately.

TGF-ß1 stimulates aromatase expression and estradiol production through SMAD2 and ERK1/2 signaling pathways in human granulosa-lutein cells.

Estradiol (E2), one of the main steroid hormones secreted by the ovaries, plays an important role in maintaining normal female reproductive function. Ovarian granulosa cells are the main source of E2 production because these cells express aromatase, which is encoded by the CYP19A1 gene and catalyzes the final step in E2 biosynthesis from androgens. Transforming growth factor-beta 1 (TGF-ß1) and its receptors are expressed in human granulosa cells, and TGF-ß1 expression can be detected in human follicular fluid. To date, TGF-ß1 has been shown to regulate various ovarian functions. However, whether aromatase can be regulated by TGF-ß1 in human granulosa cells has not been determined. In the present study, we demonstrate that TGF-ß1 stimulates aromatase expression in primary human granulosa-lutein cells and in the human ovarian granulose-like tumor cell line, KGN. We used pharmacological inhibitors and small interfering RNA-mediated knockdown approaches to reveal that the SMAD2 and ERK1/2 signaling pathways are involved in TGF-ß1-induced aromatase expression and E2 production. These results not only provide important insights into the molecular mechanisms that mediate TGF-ß1-induced aromatase expression and E2 production in human granulosa cells but also increase the understanding of the normal physiological roles of TGF-ß1 in the ovary.

Association between human SHBG gene polymorphisms and risk of PCOS: a meta-analysis.

Sex hormone-binding globulin (SHBG) is a plasma glycoprotein that binds androgens and oestrogens, and regulates their bioavailability to target tissues. To date, several human SHBG gene polymorphisms have been identified. Polycystic ovary syndrome (PCOS) is one of the most common endocrine disorders, and hyperandrogenism has been considered to be a hallmark of PCOS. Many studies have examined the association between SHBG gene polymorphisms and PCOS risk, but the results have been inconclusive or inconsistent. Therefore, the aim of this meta-analysis was to investigate whether SHBG gene polymorphisms are associated with risk of PCOS. Twelve studies were included, involving 4733 participants: 2271 patients with PCOS and 2462 control participants. The results revealed that SHBG polymorphism of eight or more (TAAAA)n pentanucleotide repeats (rs35785886) was associated with PCOS risk (odds ratio [OR] = 1.24, 95% confidence interval [CI] = 1.06, 1.44, Z = 2.77, P = 0.006) and low serum SHBG concentrations in women with PCOS (standardized mean difference = -0.83, 95% CI = -1.54, -0.12, Z = 2.30, P = 0.02). Other SHBG gene polymorphisms (rs6259, rs6257, rs727428 and rs1799941) were not significantly associated with either PCOS risk or serum SHBG concentrations. These findings suggest that the presence of a polymorphism of eight or more SHBG (TAAAA)n may be a predictive factor for the risk of PCOS.

Melatonin stimulates aromatase expression and estradiol production in human granulosa-lutein cells: relevance for high serum estradiol levels in patients with ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is one of the most life-threatening and potentially fatal complications associated with controlled ovarian hyperstimulation (COH) during in vitro fertilization (IVF) treatment. Although the pathogenesis of OHSS remains unclear, elevated serum estradiol (E2) levels before human chorionic gonadotropin (hCG) administration are associated with the risk of OHSS. The pineal hormone melatonin and its receptors are expressed in human granulosa cells and have been shown to stimulate E2 production. However, the effect of melatonin on the expression of aromatase, an enzyme responsible for a key step in the biosynthesis of E2, in human granulosa cells remains to be determined. Here, we demonstrate that melatonin upregulates aromatase expression in primary cultured human granulosa-lutein (hGL) cells through the melatonin receptor-mediated PKA-CREB pathway. Using a mouse model of OHSS, we demonstrate that administration of the melatonin receptor inhibitor luzindole inhibits the development of OHSS. In addition, the expression of ovarian aromatase and serum E2 levels are upregulated in OHSS mice compared to control mice, but this upregulation is attenuated by inhibition of the function of melatonin. Moreover, clinical results reveal that aromatase expression levels are upregulated in hGL cells from OHSS patients. Melatonin and E2 levels in the follicular fluid are significantly higher in OHSS patients than in non-OHSS patients. Furthermore, melatonin levels are positively correlated with E2 levels in follicular fluid. This study helps to elucidate the mechanisms mediating the expression of aromatase in hGL cells and provides a potential mechanism explaining the high E2 levels in patients with OHSS.

Blastocyst-stage embryos provide better frozen-thawed embryo transfer outcomes for young patients with previous fresh embryo transfer failure.

Older patients or patients with a reduced ovarian response have a low number of embryos, which limits the opportunity for embryo selection. However, for young patients undergoing frozen-thawed embryo transfer (ET), it remains unclear whether embryo stage affects pregnancy outcomes. In the present study, a total of 2952 patients undergoing their first frozen-thawed ET were divided into two groups: patients who had experienced one failed fresh ET (Group A) and patients who had not received fresh ET because of the high risk of ovarian hyperstimulation syndrome (OHSS) (Group B). Our results show that Group B patients had a significantly higher clinical pregnancy rate (CPR) and live birth rate (LBR) than Group A patients. However, Group A patients who underwent blastocyst-stage frozen-thawed ET had a significantly higher CPR and LBR and a lower ectopic pregnancy rate (ePR) than did those who underwent cleavage-stage frozen-thawed ET. In Group B, CPR, ePR, LBR and spontaneous abortion rate (sAR) were similar with blastocyst-stage and cleavage-stage frozen-thawed ET. These results suggest that blastocyst-stage frozen-thawed ET is more appropriate for young patients who had previously undergone one failed fresh ET cycle.

MicroRNA-214 protects L6 skeletal myoblasts against hydrogen peroxide-induced apoptosis.

MicroRNAs (miRNAs) have been reported as key gene regulators, and they control many fundamental biological processes. Previously, we demonstrated that miR-214 had a protective effect against myocardial apoptosis and myocardial fibrosis. In this study, we sought to investigate the expression of miR-214 in L6 skeletal myoblast (SKM), the regulatory effect of miR-214 on hydrogen peroxide (H2O2) induced cell apoptosis and the underlying mechanisms of the antiapoptotic effect. MiR-214 expression was up-regulated by H2O2 in a dose and time-dependent manner in L6 SKMs. To investigate the regulatory effects of miR-214 on L6 SKM, both gain-of-function and loss-of-function approaches were applied. The results showed that miR-214 improved cell survival and inhibited cell apoptosis, and blockage of miR-214 abrogated the protective effect on cell survival and resistance to apoptosis. Phosphatase and tensin homolog (PTEN) was negatively regulated by miR-214, and PTEN inhibitor obviously reversed the effect of miR-214 blockage on enhancing cell apoptosis. In addition, miR-214 up-regulated antiapoptotic protein Bcl-2, down-regulated proapoptotic protein Bax, prevented release of cytochrome c and inhibited caspase-3 activation. In summary, H2O2-induced injury increases miR-214 expression in L6 SKM, and miR-214 contributes to the protection of L6 SKM against apoptosis via lowering PTEN and subsequently inhibiting the mitochondrial-mediated caspase-dependent apoptotic signaling pathway.