| PurposePolycystic ovarian syndrome(PCOS)is a metabolic disease and reproductive disorder related to female hormonal imbalance worldwide.However,the specific pathogenesis of PCOS remains unclear.miR-483,a member of the miRNA family,is abnormally expressed in PCOS patients and can regulate PCOS.However,its role in PCOS is not yet clear.This article aims to observe the expression of miR-483 and IGF1 in the ovarian cortex of PCOS patients,to study the interaction between miR-483,IGF1 and insulin and the effects of the three on human ovarian granulosa cell line KGN,revealing that miR-483 is The role of PCOS in the development of PCOS and its underlying molecular mechanisms provide a potential molecular marker for the study of the mechanism of PCOS and seek new targets for the diagnosis and treatment of PCOS.Materials and methods1.Expression of miR-483 in PCOS Ovarian Cortex and Its Effect on KGN Cells Ovarian cortex tissue of 20 PCOS patients and ovarian cortical tissue of 20 non-PCOS patients were collected.The expression of miR-483 was detected by qRT-PCR.The effect of miR-483 aberrant expression on the activity and colony formation of human ovarian granulosa cell line KGN cells was examined by MTT assay and colony formation assay.qRT-PCR,Western blotting,and flow cytometry were used to detect the effect of abnormal expression of miR-483 on the mRNA and protein expression levels of the cell cycle-associated regulatory factors CCNB1,CCND1,and CDK2,and the cell cycle.2.The effect of IGF1 on KGN cells The expression of IGF1 in ovarian cortex tissue was detected by qRT-PCR and Western blotting.The online database predicts that IGF1 is the target gene of miR-483,and the targeting relationship between miR-483 and IGF1 was verified by luciferase reporter assay.The effect of abnormal expression of miR-483 on the expression of IGF1 was examined by qRT-PCR and Western blotting.The effect of abnormal expression of IGF1 on KGN cell activity and colony formation was examined using MTT assay and colony formation assay.3.Effect of insulin on KGN cells KGN cells were treated with different concentrations of insulin such as 0,1,10,and 100 ng/mL,and the expression levels of miR-483 and IGF1 mRNA in KGN cells were detected by qRT-PCR.KGN cells were treated with 100 ng/mL,and KGN cell activity and colony formation rate after 24,48,and 72 hours of insulin treatment were measured by MTT assay.Results1.The expression of miR-483 in ovarian cortex of PCOS and its effect on KGN cells The expression of miR-483 in ovarian cortex of PCOS patients was significantly decreased(P<0.001).Overexpression of miR-483 significantly inhibited the activity of KGN cells and the formation of colonies,especially at 24 hours and 72 hours after transfection(P<0.05).The reverse of miR-483 knockdown significantly promoted the proportion of S phase and G2/M phase and decreased the ratio of G0/G1 phase(P<0.01 or P<0.05).Overexpression of miR-483 significantly decreased the mRNA and protein expression levels of CCNB1,CCND1,and CDK2(P<0.01 or P<0.05),while knockdown of miR-483 promoted the mRNA and protein of these three cell cycle-related factors Expression level(P<0.05).2.Effect of IGF1 in KGN cells IGF1 expression was upregulated in ovarian cortical tissue of PCOS patients.IGF1 is the target gene for miR-483.Overexpression of miR-483 could inhibit the luciferase activity of wild-type IGF13’UTR,while there was no significant change in the mutant IGF 1 3’UTR.Overexpression of miR-483 significantly inhibited the expression of IGF1 mRNA and protein(P<0.05 or P<0.01).Overexpression of IGF1 significantly promoted the activity and colony formation of KGN cells,whereas IGF1 knockdown was the opposite(P<0.05).Over-expression of miR-483 inhibited the expression of IGF1(P<0.01)and inhibited the activity of KGN cells(P<0.05).Over-expression of miR-483 and IGF1 reversed down-regulation of miR-483-induced IGF1 expression and inhibition of cellular activity(P<0.05).3.Effect of insulin on KGN cells Insulin significantly inhibited the expression of miR-483 in KGN cells and promoted the expression of IGF1 mRNA in a dose-dependent manner(P<0.05,P<0.01 or P<0.001).Insulin significantly promoted the activity of KGN cells in a time-dependent manner(P<0.05 or P<0.01).The number of insulin-treated KGN colonies increased significantly(P<0.01).Conclusions1.miR-483 is downregulated in ovarian cortical tissue of PCOS.miR-483 can inhibit KGN cell proliferation and induce cell cycle arrest.2.IGF1 is highly expressed in ovarian cortical tissue of PCOS.IGF1 is a miR-483 target gene and miR-483 can inhibit the proliferation of KGN cells by targeting IGF 1.3.Insulin significantly inhibits miR-483,promotes IGF1 expression,and promotes KGN cell proliferation. |
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