| PurposePolycystic ovarian syndrome,PCOS)is a common reproductive endocrine disease in women of childbearing age.One of its important clinical characteristics is follicular dysplasia and ovulation disorders.Some patients are accompanied by insulin resistance and have a high risk of type 2 diabetes in the future.PCOS affects the fertility and health condition of women of childbearing age seriously.The cause of PCOS is still unclear.Ovarian Granulosa cells(GCs)are closely related to abnormal follicular development and various lesions in PCOS patients.They play an important role in follicular growth and development.Understanding the pathogenesis of ovarian granulosa cells dysfunction in PCOS may provide clues for identifying new diagnostic and therapeutic biomarkers.MicroRNA(miRNA)which abundant in ovarian tissue and follicular fluid is a special single-stranded small molecule RNA.The overexpression or down-regulation of miRNA participates in important life activities such as cell proliferation,differentiation and apoptosis by regulating gene expression,suggesting that miRNA may be related to the occurrence of PCOS.The abnormal expression of miRNAs in ovarian granulosa cells of PCOS patients and the mechanism of miRNAs regulating the pathogenesis of PCOS need further research.The research intends to clarify the pathogenesis of PCOS and provide theoretical basis for the diagnosis and treatment of PCOS by studying the biological function of miRNAs with abnormal expression in granulosa cells.Materials and methods1.Detection and target prediction of aberrantly expressed miRNAs in PCOS granulosa cells.From November 2015 to February 2018,46 PCOS patients and 32 healthy women were selected from the Department of Reproductive Medicine,the Second Affiliated Hospital of Zhengzhou University.Follicular fluid of PCOS patients and healthy control group were collected and granulosa cells were extracted.RT-qPCR was used to detect the expression differences of six candidate miRNAs(miR-33b,miR-34c,miR-106,miR-142,miR-193 and miR-423)in granulosa cells of PCOS patients and healthy controls.Online target prediction software was used to predict the target of aberrant expression of miRNAs in PCOS.Then the target gene reporter plasmid was constructed,and the predicted targets were verified by luciferase analysis and western blot.We detected the expression of TGF-?1 in the culture medium of PCOS patients and healthy controls by ELISA,and detected the effect of TGF-?1 on the expression of mir-33b,mir-142,mir-423,TGFBR1 and Smad7 in the granulosa cells by RT-qPCR.The effect of miRNAs mixture on expression of TGF-beta signaling pathway-related proteins(TGFBR1,SMAD2,SMAD3,SMAD7,CDKN1A and CDKN2B)was detected by western blotting.Finally,to further understand the pathophysiology of patients with polycystic ovary syndrome,we examined the protein levels of SMAD7 and TGFBR1 in granulosa cells from PCOS patients and healthy controls using immunoblotting.2.Biological functions of miR-142,miR-33b and miR-423 in granulosa cells.Human primary granulosa cells were transfected with miR-142 mimics,miR-33b mimics and miR-423 inhibitor for 48 h,then treated with TGF-?1 for 48 h.Cell viability was detected by MTT,apoptosis was detected by Annexin V-APC/PI double staining,and cell cycle was detected by flow cytometry.Results1.Detection and target prediction of aberrantly expressed miRNAs in PCOS granulosa cells.The expression of miR-142and miR-33b was up-regulated in GCs from PCOS patients(p<0.05),while the expression of miR-423 was down-regulated in GCs from PCOS patients(p<0.05).There was no significant difference in the expression of the remaining three microRNAs between the two groups.The target genes of microRNAs were predicted by on-line prediction software.It was found that these three microRNAs were all related to the TGF-beta signaling pathway.The target of miR-142 and miR-33b was TGFBR1,and the target of miR-423 was SMAD7.Dual luciferase reporter gene detection and protein immunoblotting were further used to validate the target predicted by the software.Compared with healthy controls,the expression of TGF-?1 in PCOS group was significantly reduced.TGF-?1 can significantly reduce the expression levels of miR-142 and miR-33b,increase the expression level of miR-423,and is time-and dose-dependent.In addition,the expression level of TGF-BR1 was significantly increased after treatment with TGF-?1 on granulosa cells,while the expression level of SMAD7 was significantly decreased.The level of TGFBR1 was decreased and the level of SMAD7 was elevated in primary GCs treated with a mixture of miRNAs.The levels of p-SMAD2 and p-SMAD3 in cells treated with the miRNAs mixture were lower when treated with TGF-?1.The expression levels of CDKN1A and CDKN2B mRNA were significantly decreased in cells treated with the mixture of miRNAs(p<0.01),while the expression level of c-Myc was significantly increased(p<0.01).In the granulosa cells of PCOS patients,the level of SMAD7 protein was increased and the level of TGFBR1 protein was decreased compared with the control.2.Biological functions of miR-33b,miR-142 and miR-423 in granulosa cells The relative cell viability of the miR-142 mimic,miR-33b mimic and miR-423 inhibitor mixtures group was significantly higher than that of the scramble control group(p<0.01).The apoptosis rate of the mixtures group was significantly lower than that of the scramble control group(p<0.05).Compared with the control group,the G0/G1 phase cells of the miR-142 mimic,miR-33b mimic and miR-423 inhibitor mixtures group were significantly decreased(p<0.05),and the S phase cells were significantly increased(p<0.01).The percentage of cells in the G2/M phase was not significantly different between the two groups.Conclusions1.Detection and target prediction of aberrantly expressed miRNAs in PCOS granulosa cellsCompared with the control group,the expression of miR-142 and miR-33b in GCs of PCOS patients was up-regulated,while the expression of miR-423 was down-regulated.The targets of these three miRNAs are involved in the TGF-?signaling pathway:the target of miR-142 and miR-33b was TGFBR1,the target of miR-423 was SMAD7.The expression of TGF-?1 in granulosa cells of PCOS patients was significantly reduced,and TGF-?1 has a negative feedback regulating effect on miR-33b,miR-142 and miR-423.MiR-142 mimics,miR-33b mimics and miR-423 inhibitor can block TGF-? signal pathway in granulosa cells.In PCOS patients,SMAD7 protein is increased in granulosa cells,while TGFBR1 protein is decreased.2.Biological functions of miR-33b,miR-142 and miR-423 in granulosa cells MiR-142 mimic,miR-33b mimic and miR-423 inhibitor have the ability to promote granulosa cell proliferation and the ability to inhibit granulosa cell apoptosis.Full text conclusionsMiR-142,miR-33b and miR-423 were abnormally expressed in granulosa cells of PCOS patients.They were involved in the development of PCOS by targeting TGFBR1 and SMAD7 to affect the biological functions of granulosa cell proliferation,apoptosis and cell cycle. |
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