Expression Of MiRNA-21-3p In The Polycystic Ovary Syndromeand Their Related Functions Study

Objective:To detect the differential expression of miRNA-21-3p in polycystic ovary syndrome(PCOS).To investigate the regulation of miRNA-21-3p on ovarian granulosa cells(Gcs)function.Materials and Methods:Rats were studied in this research.Establishment of PCOS rat model with letrozole.Measured the estrus cycle by continous dection of vaginal smear,and the morphology of the ovaries was observed by HE staining.RT-PCR was used to detect the expression of miRNA-21-3p in the ovary.Isolated and incubated primary rat ovarian granulosa cells,and than explore its growth rate and environment,Immunofluorescence experiments to evaluate whether the primary rat ovarian granulosa cells were isolated successfully or not.Transfaction miRNA21-3p mimic in primary rat ovarian granulosa cells,and than test the activity of granulosa cells.Transfaction miRNA21-3p mimic /inhibitor in primary rat ovarian granulosa cells again.The expression of proliferation-related gene PCNA,CDK2,CCNE1 and apoptosis-related gene Bax,Bcl-2,caspase-3 in granulosa cells were detected by RT-PCR when 48 hours later.Flow cytometry performed to evaluate cell apoptosis rates.All the datas were analyzed by SPSS22.0.Results:1.The PCOS rat model was successfully constructed.PCOS rats lost normal estrus cycle,weight of PCOS rats significantly higher than that of the normal group,appeared sex hormone disorder,and ovarian polycystic morphology.2.The expression of miRNA-21-3p in ovaries from rat model of PCOS was lower than that of the normal rats(p<0.05).3.Immunofluorescence experiments showed that the primary rat ovarian granulosa cells were isolated successfully.Granulosa cells can be uesd for study.4.Transfaction miRNA-21-3p(mimic)in primary rat ovarian granulosa cells and than test the activity of granulosa cells,the results showed that over-expression of miRNA-21-3p significantly inhibit granulosa cells activity when compared with the control group(p<0.05).5.Transfaction miRNA-21-3p mimic/inhibitor in primary rat ovarian granulosa cells,and than test the expression of PCNA?CDK2?CCNE1 witch is considered to be a proliferation re lated gene;the results showed that over-expression of miRNA-21-3p can significantly reduce the expression of PCNA?CDK2?CCNE1(p<0.05),inhibit miRNA-21-3p expression can promot the expression of PCNA,CDK2,CCNE1(p<0.05).6.Transfaction miRNA-21-3p mimic/inhibitor in primary rat ovarian granulosa cells,and than test the expression of Bax?Bcl-2?caspase-3 witch is considered to be a apoptosis related gene;The results showed that over-expression of miRNA-21-3p can significantly promote the expression of Bax,caspase-3,reduce the expression of Bcl-2(p<0.05),inhibit miRNA-21-3p expression can reduce the expression of Bax,caspase-3,promote the expression of Bcl-2(p<0.05).7.Transfaction miRNA-21-3p mimic/inhibitor in primary rat ovarian granulosa cells,and than test the apoptosis rates of granulosa cells by flow cytometry.The results showed that over-expression of miRNA-21-3p can significantly inceased the apoptosis rates of granulosa cells(p<0.05),inhibit miRNA-21-3p expression can significantly reduced the apoptosis rates of granulosa cells(p<0.05).Conclusion:The PCOS rat model was successfully constructed.The expression of miRNA-21-3p in PCOS rats ovary was lower than that of normal control rats.MiRNA-21-3p can significantly inhibit granulosa cells activity,downregulate the expression of PCNA,CCNE1,CDK,Bcl-2,upregulate the expression of Bax,caspase-3,supresse the proliferation of granulosa cells and promote the apoptosis of ovarian granulosa cells.