| ObjectivePolycystic ovary syndrome(PCOS)is the most common reproductive endocrine disease in woman of childbearing age,ofen accompanied with metabolic disorder,nearly 80 % of patients with PCOS surffered different degrees of insulin resistance(polycystic ovary syndrome with insulin resistance,PCOS-IR).Our group used high-throughput gene sequencing technology to screen out miR-136-3p,which is highly expressed in ovarian tissue of SD rats model of PCOS-IR while comparing with control group.Then we predicted its potential target gene insulin receptor substrate-1(IRS-1),preliminarily clarificated the regulation of between miR-136-3p and IRS-1 and its downstream pathway phosphatidylinositol-3 kinase/protein kinase B(PI3K/AKT).We transfected human granulosa-like tumor cell(kartogenin,KGN cell)and human renal epithelial cell(293T cell)with miR-136-3p,in order to further investigate the molecular mechanism of miR-136-3p regulates the IRS-1/PI3K/AKT signaling pathway involved in PCOS-IR.Method(1)High-throughput gene sequencing technology was used to analyze the differential microRNAs(miRNAs)expression profile in the ovaries of PCOS-IR rats and control rats group.Finally,bioinformatics method was used to screen out miRNAs,which is high specific expressed in PCOS-IR,and its biological potential target genes.(2)Expression of miR-136-3p and its relationship with IRS-1/PI3K/AKT pathway: 1)We analyzed the mRNA expression of miR-136-3p and IRS-1in the ovaries of two rats groups by using real time polymerase chain reaction(RT-PCR);2)Western blot analysis was used to analyze the protein expression of IRS-1,PI3 K,AKT,phosphorylated phosphoinositide 3-kinase(pPI3K)and phosphorylated protein kinase B(pAKT)in the ovaries of two rats groups.(3)To observe the effect of miR-136-3p and its different concentrations on KGN cells and 293 T cells: miR-136-3p mimic and miR-136-3p inhibitor transfection reagent and different gradient concentrations were designed to transfect KGN cells and 293 T cells in order to observe the effects of miR-136-3p enhancement or inhibition and its different concentrations on the mRNA expression of IRS-1 and the protein expression of IRS-1,PI3 K,AKT,pPI3 K,pAKT in KGN cells and 293 T cells.(4)To observe the protein interaction between IRS-1/PI3K/AKT pathway: the direct interaction of protein between IRS-1,pPI3 K and pAKT was detected by co-immunoprecipitation(COIP)magnetic bead assay.Results(1)High-throughput gene sequencing results of two rats groups showed that miR-136-3p in the PCOS-IR group was significantly higher than that in the control group.We used bioinformatics techniques to predicted its target gene IRS-1,related GO analysis and KEGG analysis showed that it is closely related with PI3K/AKT pathway.(2)RT-PCR experiments showed that: 1)The mRNA expression of miR-136-3p in the ovarian tissue of PCOS-IR group was significantly higher than that in the control group,and the mRNA expression of IRS-1was significantly lower than that in the control group(P<0.05 in both of the cell lines);2)After 48 hours of miR-136-3p mimic transfection of KGN cells and 293 T cells,the mRNA expression of IRS-1 was significantly lower than that of the control group.And the results of the inhibitor group were opposite(P<0.05 in both of the cell lines).(3)Western blot results showed that: 1)The expression of IRS-1,pPI3 K and pAKT in the PCOS-IR model group were significantly lower than that in the control group(P<0.05);2)After 48 hours of miR-136-3p mimic transfection of KGN cells and 293 T cells,the protein expressions of IRS-1,pPI3 K and pAkt were significantly lower than that in the control group,and the results of the inhibitor group were opposite(P<0.05 in both of the cell lines);3)The protein expression of IRS-1,pPI3 K and pAKT was significantly lower than that in the control group after transfecting different gradient concentrations of miR-136-3p mimic with KGN cells and 293 T cells for 48 hours.And as the concentration increased,the protein expression decreased significantly,while the results of the inhibitor group are reversed(P<0.05 in both of the cell lines).(4)The results of COIP experiment showed that there was a direct interaction between IRS-1 and pPI3 K in the protein level.Conclusion(1)IRS-1/PI3K/AKT signaling pathway is one of the pathways for.miR-136-3p participates in the IR mechanism of ovaries of PCOS-IR rat group and granulosa cells(2)IRS-1 directly interacts with pPI3 K in protein level,and together with PI3K/AKT constitute an insulin molecular signal transduction pathway. |
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