| Background and ObjectiveThe incidence of polycystic ovary syndrome(PCOS)is increasing year by year.Many factors,such as heredity,environment and lifestyle,are considered to be the important causes of PCOS.The clinical manifestations of P COS are highly heterogeneous.The diagnosis and treatment of PCOS are still controversial.There is no specific and radical treatment in clinic,mainly symptomatic treatment.At the same time,long-term health management is needed,especially the prevention and treatment of long-term complications and metabolic syndrome.Existing studies suggest that metabolic disorder caused by hyperandrogenism is one of the main characteristics of PCOS,and this metabolic and endocrine disorder may affect women's life.As a new androgen receptor regulator,a kind of long non coding RNA,C-Terminal binding protein 1 antisense(LncRNA CTBP1-AS)has been proved to be associated with the progress of hormone-dependent and androgen-resistant prostate tumors.A few studies have found that lncRNAA CTBP1-AS is highly expressed in small samples of PCOS patients and is associated with the risk of PCOS,but its specific function and mechanism have not been clearly reported.Therefore,in this study,peripheral blood mononuclear cells,follicular fluid granulosa cells and ovarian granulosa cell lines from patients with PCOS were taken as the research objects.The function,molecular mechanism and clinical significance of lncRNAA CTBP1-AS in the occurrence and development of PCOS were systematically studied from the perspective of gene transcription level,regulation of noncoding RNA,such as miRNA,expression and other indicators.The research provides new insights into the pathogenesis of PCOS,and explores new biomarkers and therapeutic targets for individualized clinical diagnosis and treatment of PCOS.Methods and results1.The expression of lncRNAA CTBP1-AS in peripheral blood mononuclear cells of PCOS and control group were detected by RT-qPCR.It was found that the average level of lncRNAA CTBP1-AS in peripheral blood mononuclear cells of PCOS group was higher than that of control group(P < 0.05).2.Further grouping the subtypes and clinical characteristics of PCOS,we found that the expression of lncRNAA CTBP1-AS in complete PCOS type,hyperandrogenism + insulin resistance group and hyperandrogenism group increased significantly(P < 0.05).The average expression level of lncRNAA CTBP1-AS in ovulation type,non-hyperandrogenism type,non-hyperandrogenism + insulin resistance group were higher than that in control group,but there was no statistically difference(P > 0.05);the average expression level of lncRNAA CTBP1-AS in classical type and insulin resistance group were not significantly different from that in control group(P > 0.05).3.Pearson or Spearman correlation analysis was used to analyze the expression of lncRNAA CTBP1-AS and clinical parameters.The results showed that t he expression of lncRNAA CTBP1-AS was positively correlated with the levels of antral follicles and androgen in all subjects(P < 0.05).4.The expression level of lncRNAA CTBP1-AS in granulosa cells of follicular fluid in PCOS and control group were detected by RT-qPCR.The average expression level of lncRNAA CTBP1-AS in granulosa cells of PCOS patients was higher than that of control(P < 0.05);Pearson or Spearman correlation anal ysis showed that lncRNAA CTBP1-AS level was positively correlated with menstrual cycle and negatively correlated with age and total Gn(P < 0.05).5.CCK8 and flow cytometry were used to detect the effect of overexpression of lncRNAA CTBP1-AS on the function of granulosa cells.The results showed that the apoptosis of KGN cells increased after overexpression of lncRNAA CTBP1-AS,especially the proportion of late apoptotic cells increased(P < 0.05).The ability of cell proliferation of KGN cells was reduced,but the difference was not statistically significant(P > 0.05).The results of cell migration assay showed that the migrat ion ability of KGN cells was significantly decreased after overexpression of lncRNAA CTBP1-AS(P < 0.05),while there was no significant difference in the percentage of cells in G0-G1,S and G2-M phases of KGN cells (P > 0.05).6.High throughput sequencing was used to detect the changes of gene expression profiles of miRNAs and mRNAs in KGN cells after overexpression of lncRNAA CTBP1-AS.A total of 28 differentially expressed miRNAs were obtained from the overexpression group and the control group,of which 12 were up-regulated and 16 were down-regulated in the OE group.A total of 282 differentially expressed genes were obtained from the RNA sequencing results,of which 166 were up-regulated and 116 were down-regulated.7.GO enrichment analysis of target genes of differentially expressed miRNAs showed that these genes were mainly related to the regulation of transcription of RNA polymerase II promoter,protein phosphorylation/autophosphorylation,regulation of meiotic recombination,regulation of exocytosis,regulation of Ras protein signal transduction;GO enrichment analysis of RNA sequencing differentially expressed genes showed that these genes were mainly related to the regulation of cytokines,leukocyte activation,leukocyte migration and chemotaxis,inflammatory response,cell activation.8.KEGG pathway analysis of target genes of differentially expressed miRNAs suggest ed that these genes were mainly involved in endocrine and metabolic diseases,immune system,cell growth and apoptosis in cell process,signal transduction and other pathways;KEGG pathway analysis of target genes for differentially expressed mRNAs suggest ed that these genes were mainly involved in Cytokine-cytokine-receptor interaction,protein and carbohydrate digestion and absorption,PI3K-Akt signaling pathway,MAPK signaling pathway and other related signal transduction pathways.9.The GO enrichment and KEGG intersection analysis of differentially expressed genes(miRNAs and mRNA)and the construction of co-expression regulatory network were carried out.The results showed that 44 common items(P < 0.05)and 7 common items(P < 0.01)were found,which were mainly involved in negative regulation of cell proliferation,positive regulation of T cell activation,positive regulation of MAPK cascade,positive regulation of apoptotic process,and cell migration.Conclusions1.LncRNA CTBP1-AS were highly expressed in peripheral blood mononuclear cells and granulosa cells of follicular fluid in patients with polycystic ovary syndrome,and correlated with clinical parameters such as sinus follicles and androgen levels,suggesting that lncRNAA CTBP1-AS may be involved in the occurrence and progress of PCOS.2.The results of cell function experiment showed that lncRNAA CTBP1-AS could obviously promote apoptosis and inhibit migration of granulosa cells,suggesting that lncRNAA CTBP1-AS may cause pathological state of PCOS,such as follicular development retardation,ovulation disorder and hyperandrogenism,by altering the function of ovarian granulosa cells.3.High-throughput sequencing revealed changes in the expression profiles of some miRNAs and mRNAs in granulosa cells after overexpression of lncRNAA CTBP1-AS.The co-expression regulatory network suggested that lncRNAA CTBP1-AS may affect the expression of multiple genes through PI3K-Akt signaling pathway,MAPK signaling pathway,or Ce RNA mechanism,which affect cell proliferation,apoptosis and play a role in the endocrine immune system,thus participate in the pathological process of PCOS. |
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