Identification Of Differentially Expressed MiRNAs In Granulosa Cells Of Polycystic Ovary Syndrome And Its Clinical Significance

Objective:Polycystic ovary syndrome(PCOS)is an endocrine disorder that affects about 10% of women of childbearing age.Reproductive dysfunction is a common clinical manifestation in patients with PCOS.Granulosa cells play an important role in the development of oocyt e and the maintenance of oocyte microenvironment,while its dysfunction leads to the abno rmal development of follicular in patients with PCOS.MicroRNAs(miRNAs)are small no ncoding RNAs(22?24 nucleotide),that degrade/inhibit target gene translation by inducing degradation of target gene or binding to the 3'nontranscriptional region(UTRs)of the targ et gene.Studies have shown that abnormal expression of miRNAs was involved in primord ial follicular formation,follicular recruitment and selection,follicular atresia,cumulus celloocyte interactions,and granulosa cells function.However,its mechanism is not fully clear.The aim of the study is to screen differentially expressed miRNAs in granulosa cells from PCOS.Then investigate the biological functions and regulatory mechanisms and analyze their potential clinical significance.Methods:1.In this study,Gene microarray data(GSE84376)from the GEO(Gene Expression Omnibus)database were firstly used to screen out differential miRNA related to PCOS.Target gene prediction software was used to predict the target genes of these differential miRNAs,and functional enrichment analysis was carried out on them.Then,the STRING online tool and Cytoscape software were used to construct the protein interaction network(PPI)to find the hub genes,and the miRNA-hub gene regulation network was further constructed.Finally,FunRich software was used to enrich and analyze the potential transcription factors of differentially expressed miRNAs target genes.2.Clinical data and granulosa cells were collected to verify differentially expressed miRNA by RT-qPCR,and the correlation between miRNA and clinical collection indicators was analyzed.Results:1.After gene microarray screening,a total of seven differentially up-regulated miRNAs were screened in this study.We have enriched the biological function associated with PCOS,including "cellular response to nitrogen compound"(GO: 1901699),"blood vessel development"(GO: 0001568),"positive regulation of hydrolase activity "(GO: 0051345),"Axon guidance"(hsa04360),"regulation of calcium ion transport"(GO: 0051924),“chloride transmembrane transport”(GO: 1902476).Through PPI network and miRNA hub gene regulation network construction,a total of 32 target genes were identified as hub genes,and these key genes were mainly regulated by hsa-miR-3135 b,hsa-miR-1225-5p,hsa-miR-1587,hsa-miR-3188,hsa-miR-4433-3p and hsa-miR-4417.In the enrichment analysis of transcription factors of different miRNA target genes,we found the ten most significant transcription factors,and the highest enrichment degree of Sp1.2.Ovarian granulosa cells were verified by RT-qPCR,and six miRNAs were upregulated,among which the difference between hsa-miR-3135 b and hsa-miR-3188 was statistically significant(P< 0.05).The results of correlation analysis between hsa-miR-3135 b expression and clinical data showed that hsa-miR-3135 b was positively correlated with the number of oocytes retrieved(r=0.450,P<0.001),and negatively correlated with the fertilization rate and cleavage rate(r=-0.280,P=0.016;r =-0.232,P=0.049,respectively).Conclusion:In our research,the bioinformatics methods were used for the first time to screen and prove that hsa-miR-3135 b was highly expressed in granulosa cells of PCOS patients,and it was correlated with number of oocytes retrieved,fertilization and cleavage,suggesting that the abnormal expression of miR-3135 b may affect the development of oocytes,providing a theoretical basis for the study of PCOS follicular development disorders.