Shuxiong Chen

Differential Expression of microRNAs in the Ovaries from Letrozole-Induced Rat Model of Polycystic Ovary Syndrome

Polycystic ovary syndrome (PCOS) is a complex and heterogeneous endocrine disorder. To understand the pathogenesis of PCOS, we established rat models of PCOS induced by letrozole and employed deep sequencing to screen the differential expression of microRNAs (miRNAs) in PCOS rats and control rats. We observed vaginal smear and detected ovarian pathological alteration and hormone level changes in PCOS rats. Deep sequencing showed that a total of 129 miRNAs were differentially expressed in the ovaries from letrozole-induced rat model compared with the control, including 49 miRNAs upregulated and 80 miRNAs downregulated. Furthermore, the differential expression of miR-201-5p, miR-34b-5p, miR-141-3p, and miR-200a-3p were confirmed by real-time polymerase chain reaction. Bioinformatic analysis revealed that these four miRNAs were predicted to target a large set of genes with different functions. Pathway analysis supported that the miRNAs regulate oocyte meiosis, mitogen-activated protein kinase (MAPK) signaling, phosphoinositide 3-kinase/Akt (PI3K-Akt) signaling, Rap1 signaling, and Notch signaling. These data indicate that miRNAs are differentially expressed in rat PCOS model and the differentially expressed miRNA are involved in the etiology and pathophysiology of PCOS. Our findings will help identify miRNAs as novel diagnostic markers and therapeutic targets for PCOS.

Role of lncRNAs as Novel Biomarkers and Therapeutic Targets in Ovarian Cancer.

Ovarian cancer (OC) is the leading cause of death among all gynecological malignancies in the world and its underlying mechanism is still unclear. Compared with research on microRNAs, research on long non-coding RNAs (lncRNAs) is still in its infancy. Studies in recent years have demonstrated that lncRNAs exhibit multiple biological functions in various stages of OC development. In this review, we conclude that lncRNAs are closely involved in the pathogenesis of OC. The expression of lncRNAs indicates the early diagnosis, prognosis, and response to chemotherapy of OC. An attractive approach to treatment of OC is lncRNA small interfering RNA or acting as a plasmid targeting the expression of toxic genes, which is a novel step toward a major breakthrough in the treatment of human OC. E2-regulated lncRNA and its polymorphism, methylation, are also involved in OC. Further research efforts are needed before fully identifying, characterizing, and elucidating the actual functions of lncRNAs in OC at the molecular level and putting them into clinical practice.

MicroRNA-16 Modulates Melatonin-Induced Cell Growth in the Mouse-Derived Spermatogonia Cell Line GC-1 spg Cells by Targeting Ccnd1

ABSTRACT Melatonin exerts a range of physiological effects. However, the functional significance of melatonin in spermatogenesis and the underlying mechanisms remain unclear. MicroRNAs (miRNAs) are essential in the initiation and progression of testicular development, including spermatogenesis. Thus far, limited information is known about the role of miRNAs in melatonin-mediated spermatogenesis. In this study, the expression levels of testicular miRNA machinery genes, namely, Dgcr8 and Xpo5, were significantly increased by melatonin. The miRNA expression profile was identified in the testes of melatonin-treated mice. Ten miRNAs were significantly up-regulated, and 15 miRNAs were down-regulated. Melatonin (25 μM) enhanced cell growth and reduced apoptosis in GC-1 spg cells. Among the down-regulated miRNAs, miR-16 expression was influenced by melatonin in GC-1 spg cells. The miR-16 mimics in GC-1 spg cells significantly suppressed cell growth and promoted cell apoptosis. Conversely, transfection of the miR-16 inhibitor significantly increased cell growth and decreased cell apoptosis. The protein expression level of CCND1 (Cyclin D1) in GC-1 spg cells was decreased by the miR-16 mimics and increased by knockdown of miR-16. Moreover, bioinformatics and reporter activity analyses showed that Ccnd1 was a potential target of miR-16. These results suggested that miR-16 may function as a novel regulator of testicular functions during melatonin stimulation by targeting Ccnd1.

The interactions between nerve growth factor and gonadotrophins in bovine oviduct.

Nerve growth factor promotes the survival and differentiation of nervous cells and is thought to play an important role in the development of reproductive tissues. The aims of this work were to detect the presence of NGF and its receptor NTRK1 in bovine oviduct samples, and to investigate the regulatory interactions between NGF/NTRK1 and gonadotrophins in bovine oviduct epithelial cells. Both transcripts and proteins of NGF and NTRK1 were detected by RT-PCR and Western blotting, and the corresponding proteins were specifically immunolocalized in oviduct epithelial cells. In addition, real-time PCR experiments revealed that the levels of NGF and NTRK1 mRNA in oviduct epithelial cells treated with exogenous FSH or LH were greater than those in negative control cells (P<0.05). Similarly, treatment with NGF significantly increased the expression of FSHR and LHR in oviduct epithelial cells via its effects on NTRK1 (P<0.05). This process was suppressed by treatment with the NTRK1 inhibitor K252α. We conclude that NGF/NTRK1 may have a role in regulating the function of bovine oviducts via its interactions with gonadotrophins.

Altered expression of miRNAs in the uterus from a letrozole-induced rat PCOS model.

Polycystic ovary syndrome (PCOS) causes female subfertility with ovarian disorders and may be associated with increased rate of early-pregnancy failure. Rat PCOS models were established using letrozole to understand the uterine pathogenesis of PCOS. The differential expression of microRNAs (miRNAs) was observed in rat uterus with PCOS. After estrous cycles were disrupted, significantly abnormal ovarian morphology and hormone level were observed in rats with PCOS. A total of 148 miRNAs differentially expressed were identified in the uterus from the letrozole-induced rat model compared with the control. These miRNAs included 111 upregulated miRNAs and 37 downregulated miRNAs. The differential expression of miR-484, miR-375-3p, miR-324-5p, and miR-223-3p was further confirmed by quantitative reverse transcription polymerase chain reaction. Bioinformatic analysis showed that these four miRNAs were predicted to regulate a large number of genes with different functions. Pathway analysis supported that target genes of miRNAs were involved in insulin secretion and signaling pathways, such as wnt, AMPK, PI3K-Akt, and Ras. These data indicated that miRNAs differentially expressed in rat uterus with PCOS may be associated with PCOS pathogenesis in the uterus. Our findings can help clarify the mechanism of uterine defects in PCOS.

4‐Methylcatechol‐induced cell damage in TM4 Sertoli cells

4‐Methylcatechol (4‐MC) is one of the metabolites of quercetin, which is a potential drug for neuroprotection and tumorigenesis inhibition. This study was performed to investigate the cytotoxic effect of 4‐MC in mouse TM4 Sertoli cells. TM4 Sertoli cell viability was significantly inhibited by 4‐MC in a time‐ and dose‐dependent manner. The number of apoptotic and dead cells was significantly increased after 4‐MC treatment. Caspase 3 activity increased by prolonged exposure of TM4 Sertoli cells to 200 μM 4‐MC. The 4‐MC significantly upregulated the mRNA level of Bax gene and considerably downregulated the Bcl‐2 gene expression in a concentration‐dependent manner. Results showed that 4‐MC could induce TM4 Sertoli cell apoptosis, and the cytotoxic effect of 4‐MC on TM4 Sertoli cells may be associated with upregulated Bax gene expression, which induced caspase cascade activation.

A new insertion/deletion fragment polymorphism of inhibin-α gene associated with follicular cysts in Large White sows

Ovarian follicular cysts are anovulatory follicular structures that lead to infertility. Hormones play key roles in the formation and persistence of cysts. Inhibins are heterodimeric gonadal glycoprotein hormones that belong to the transforming growth factor-β superfamily. These hormones suppress the secretion of follicle-stimulating hormone. In this report, partial fragment of inhibin-α (INHA) subunit gene of Large White pig was detected from the genomic DNA by polymerase chain reaction. The sequence showed a 283 bp fragment insertion/deletion (I/D) polymorphism in INHA subunit gene. A total of 49 Large White sows with cystic follicles and 152 normal sows were screened for this polymorphism. The relationship of INHA I/D polymorphisms with follicular cysts was investigated. The distribution of I/D was significantly different between cystic and normal sows, thereby suggesting that the INHA subunit gene might be a potential biological marker for breeding programs in pig.

Melatonin promotes the proliferation of GC-1 spg cells by inducing metallothionein-2 expression through ERK1/2 signaling pathway activation

Synthesized by the pineal gland, melatonin is a neurohormone implicated in diverse physiological functions via several mechanisms. However, the role of melatonin in spermatogenesis and its underlying mechanisms have yet to be completely understood. In the present study, transcriptome sequencing was performed to characterize the mechanism of melatonin-induced GC-1 spg proliferation. Gene ontology (GO) enrichment and pathway analyses were also conducted to identify the signaling pathways and biological processes involved in differential mRNA expression. Results revealed 28 differential genes. Of these genes, 11 were upregulated and 17 were downregulated. Melatonin increased the expression of metallothionein-2 (Mt2), a gene that acts as a protector to sequester nonessential toxic heavy metals. Functional investigations demonstrated that Mt2 overexpression promoted the proliferation of GC-1 spg cells, but Mt2 knockdown significantly suppressed their proliferation and increased their apoptosis. Mechanistic analysis indicated that the extracellular-signal-regulated kinase 1/2 (ERK1/2) pathway participated in melatonin-promoted proliferation of GC-1 spg cells. Therefore, melatonin induces the proliferation of GC-spg 1 cells by stimulating Mt2 expression, and this process is mediated by the ERK1/2 signaling pathway.

4-Methylcatechol inhibits cell growth and testosterone production in TM3 Leydig cells by reducing mitochondrial activity.

4‐Methylcatechol (4‐MC) is a potential neuroprotective drug because it stimulates the synthesis of brain‐derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in neurons. The present study explored the effect of 4‐MC on cell growth and testosterone synthesis in the TM3 Leydig cells of mice. 4‐MC did not enhance expression of both BDNF and NGF in these cells. However, this compound significantly inhibited cell proliferation and increased the number of apoptotic cells in a dose‐dependent manner. The expression profile of Bax/Bcl‐2 gene was altered considerably, and mitochondrial activity was significantly decreased in cells. 4‐Methylcatechol also inhibited testosterone synthesis in TM3 Leydig cells. The inhibitory roles of this compound in relation to growth and testosterone synthesis in TM3 Leydig cells maybe associated with increased Bax gene expression and decreased mitochondrial activity. As a result, caspase cascade is activated.

Association of inhibin-α gene polymorphisms with follicular cysts in large white sows

Ovarian follicular cysts are anovulatory follicular structures that have been identified in sows and are known to cause infertility. The pathogenesis of follicular cysts remains poorly understood. Hormones play key roles in the formation and persistence of cysts. The hormone inhibin is a member of the TGF-β superfamily and is named for its negative regulation of FSH, another hormone that controls follicular recruitment and growth. In the present study, 48 sows with follicular cysts and 60 normal sows with no cysts were screened for mutations in the inhibin-α gene to examine the association of inhibin-α gene polymorphisms with the presence of follicular cysts. The results show that the c.-42G>A and c.3222G>A polymorphisms are significantly associated with follicular cysts and that sows with c.-42GG and c.3222GG genotypes have lower risk of developing cysts. Our findings may provide novel biological biomarkers and promising gene therapy candidates for follicular cyst formation in sows, which would greatly benefit pig breeding programs.