Expression Of MiR-let-7d-3p In Polycystic Ovary Syndrome And Studies Of Its Function And Mechanism

BackgroundPolycystic ovary syndrome(PCOS)is a common gynecological endocrine disease,which is one of the main causes of anovulatory infertility in women at childbearing age.It is clinically characterized by clinical or biochemical manifestations of excessive androgen,rare ovulation or persistent anovulation,polycystic ovarian changes,and often accompanied by insulin resistance and abnormal lipid metabolism.The pathogenesis of PCOS is complex,involving genetic,environmental and metabolic factors,but it has not been fully elucidated.Micro RNAs(miRNAs)are a kind of non-coding small RNA in eukaryotes,with a length of about 18-24 nucleotides.They exist in a variety of physiological and pathological activities in the body,such as growth,inflammation,reproduction and metabolism.Although miRNAs cannot directly encode proteins,they can bind to the 3’untranslated region of the target genes m RNA,inhibiting the translation of the m RNA,and thus play a role in regulating gene expression.The mi R-let-7 family is a member of miRNAs,which can regulate follicle development and maturity,steroid levels,and is closely related to the female reproductive system.Ovarian granulosa cell is distributed around oocyte,synthesizing a variety of sex hormones,and has an important effect on follicle development.Abnormal change in granulosa cell can cause abnormal follicle development,affecting normal ovarianfunction,and leads to the occurrence of PCOS.Mi RNAs play an important role in the biological activities of granulosa cell in patients with PCOS.More and more studies have confirmed that miRNAs are involved in the occurrence and development of PCOS.For example,miRNA-200 b and miRNA-200 c are up-regulated in granulosa cell of PCOS patients.Mi RNA-200 b and miRNA-200 c can inhibit the proliferation of human ovarian granulosa cell line KGN cell and cause abnormal proliferation of granulosa cell,which may be a potential cause of PCOS.The miRNA family is huge and numerous,and further exploration of miRNAs related to PCOS is needed to provide a new foundation for better revealing its pathogenesis.ObjectiveTo analyze the expression of miRNAs in granulosa cell of PCOS patients and find differentially expressed miRNAs in PCOS patients.To study the effect of differentially expressed miRNAs on KGN cell function,and to study the target genes and related signaling pathways of miRNAs,thus providing new insight into the pathogenesis of PCOS.Materials and methodsTwenty-three PCOS patients assisted with IVF/ICSI assisted pregnancy were collected as the experimental group,while 20 non-PCOS patients assisted with IVF /ICSI assisted pregnancy due to fallopian tubes or male factors were collected as the control group.Three cases were collected in each group,and miRNA deep sequencing analysis was performed on their granule cell using next-generation sequencing technology to obtain miRNAs expression profile.Six miRNAs with significant differences(P <0.05)in sequencing expression profile and relative high expression levels were selected,and the miRNAs obtained by sequencing were verified by clinical samples using RT-q PCR technology,then miRNAs with statistical differences were found.Then miRNA si RNA small interference treatment was performed in KGN cell to study the effect of miRNA on KGN cell function.Then use bioinformatics technology to predict and analyze miRNA target genes,and find target gene related signal pathway.After si RNA treatment in KGN cell,use dual-luciferase reporter gene experiment to detect whether miRNA directly binds to the target gene.RT-q PCR,Western Blot experimental research found out the regulation of miRNA on target genes and related signaling pathway.Results(1)Through deep sequencing and analysis of miRNAs in two groups of granulosa cell,72 miRNAs with statistic difference were obtained,including 10 up-regulated miRNAs and 62 down-regulated miRNAs.(2)The expression of mi R-let-7d-3p was significantly increased in granulosa cells of PCOS patients(P = 0.0091),while there was no statistical significance between two groups in miRNA-134-5p,miRNA-181a-5p,miRNA-328-3p,miRNA-409-3p. miRNA-493-3p expression.(3)CCK-8 experiment showed that mi R-let-7d-3p over-expression could inhibit KGN cell proliferation.(4)Mi R-let-7d-3p could directly bind to the target gene TLR4,inhibiting the expression of m RNA and protein of TLR4,and inhibit the expression of Wnt/β-catenin signaling pathway protein.Conclusion(1)The expression of mi R-let-7d-3p in granulosa cell of PCOS patients was significantly increased.(2)Mi R-let-7d-3p may mediate TLR4 gene to inhibit granulosa cell proliferation through the Wnt /β-catenin signaling pathway,and thus play a role in the pathogenesis of PCOS.