The Molecular Mechanism Of MiR-133b In Regulating Polycystic Ovary Syndrome Development

Polycystic ovary syndrome(PCOS)is one of the most common endocrine disorders in women of childbearing age,mainly characterized by rare ovulation or no ovulation,high testosterone levels,and the appearance of polycystic ovary changes under ultrasound.Its incidence rate is 5% to 11%.In recent years,polycystic ovary syndrome has become a serious disease affecting the female reproductive and health.Some pathology symptoms are thought to be related to PCOS,such as insulin resistance,hyperinsulinemia,hyperandrogenism,immune factors imbalance,changed environmental and psychological factors.However the detail mechamism of PCOS is not clear.It is known that GCs play an important role as mediators of follicular developmental potential via oocyte paracrine signals in PCOS.Micro RNAs(mi RNAs),the noncoding RNAs(22-24 nt),are involved in many cellular processes,including cell proliferation,differentiation,and death,under both physiological and pathological conditions.The important role of mi RNAs in the development and function of reproductive tissues is now becoming increasingly understood.Hundreds of mi RNAs are present in the ovary,and they are associated with the regulation of genes involved in many processes,including the cell cycle,cell growth,cell proliferation,cell differentiation,angiogenesis,steroidogenesis,and atresia.In our previous studies,miR-133b was upregulated over 30-fold in MI oocytes after IGF-1 treatment,and it was shown that miR-133b maybe involved in the growth and maturation of oocytes by regulating TAGLN2.In the present study,we investigated miR-133b to explore its functions in human granulosa cell(h GC)proliferation and apoptosis and to elucidate the underlying mechanisms.Part I The effects of miR-133b on the proliferation and apoptosis of human granulosa cells1.Aim.To investigate the detail mechanism of miR-133b on the proliferation and apoptosis of human granulosa cells.2.Methods Human granulosa cells(HGCs)were collected from the oocyte-cumulus complexes of patients undergoing ICSI,and divided them into 5 groups,control group,mi R133b-mimic group,mi R133 b mimic-negative control group,miR-133b inhibitor,mi R-133b inhibitor-negative control group,respectively.CCK-8 is used to analyse the cell proliferation in vitro.Flow cytometry was used to detect the apoptosis rate and cell cycle of human granulosa cells treated with miR-133b.QRT-PCR was used to detect the RNA levels of miR-133b,TAGLN2 and caspase-3 in the human granulosa cells.Western blot was used to detect the protein levels of TAGLN2,procaspase-3,and cleaved caspase-3 in each group.3.Results 1).Human granulosa cells cultured in vitro.However,they degenerated and died due to the lack of support from various hormones and regulatory factors in the body.Human granulosa cells proliferated very well from day 2 to day 3.Cells were in good condition and reached a peak at day 4.And then,During the culture period,the filamentous projections stretched gradually.Then,the h GCs began to degenerate and die from day 5 to day 8.Compare to day 2,the expression of miR-133b was increased(P<0.01)and the expression of TAGLN2 and caspase-3 were decreased(P<0.01)in human granulosa cells from day 2 to day 4.Compare to day 4,the expression of miR-133b was decreased(P<0.01)and the expression of TAGLN2(P<0.01)and caspase-3(P<0.05)were increased in human granulosa cells from day 5 to day 8.2).To assess the effects of miR-133b on h GC growth,human granulosa cells were transfected with a miR-133b mimic,a miR-133b mimic NC,a miR-133b inhibitor,or a miR-133b inhibitor NC.The results of the CCK-8 assay showed that proliferation of cells overexpressing miR-133b was significantly increased after transfection(P< 0.01).3).Human granulosa cells were transfected with a miR-133b mimic,a miR-133b mimic NC,a miR-133b inhibitor,a miR-133b inhibitor NC.Compared to the negative control,an apoptosis or cell cycle assay demonstrated that the overexpression of miR-133b caused h GCs to undergo apoptosis(P< 0.01)or S/G2 rest(P<0.01).4).Human granulosa cells were transfected with a miR-133b mimic,a miR-133b mimic NC,a miR-133b inhibitor or a miR-133b inhibitor NC.The results indicate that the overexpression of miR-133b decreased both the protein and m RNA levels of TAGLN2(P<0.05)and cleaved caspase-3(P<0.01)in h GCs.Furthermore,the miR-133b inhibitor enhanced the protein and m RNA expression of TAGLN2(P<0.05)and cleaved caspase-3(P<0.05)in human cumulus granulosa.Part II The effect of miR-133b on granulosa cell proliferation in polycystic ovary syndrome patients1.Aim To investigate the effect of miR-133b on the proliferation of human granulosa cells in polycystic ovary syndrome patients.2.Methods Human granulosa cells(HGCs)were collected from the oocyte-cumulus complexes of polycystic ovary syndrome patients and isolated granulosa cells for further research.CCK-8 is used to analyse the cell proliferation in vitro.QRT-PCR was used to detect the RNA levels of miR-133b and caspase-3 in the human granulosa cells.Western blot was used to detect the protein levels of cleaved caspase-3 and miR-133b in human granulosa cell.3.Results.1).Human granulosa cells from PCOS patients cultured in vitro.Cells were in good condition and reached a peak at day 3.And then,during the culture period,the filamentous projections stretched gradually.Then,the h GCs began to degenerate and completely apoptosis at day 8(P< 0.05).2).MiR-133b and caspase-3 expression showed an opposite trend between PCOS patients and male-infertility patients.Overexpression of miR-133b decreased both the protein and m RNA levels of TAGLN2 (P<0.05)and cleaved caspase-3(P<0.01).Furthermore,the miR-133b inhibitor enhanced the expression of TAGLN2(P<0.05)and cleaved caspase-3(P<0.05)in human cumulus granulosa.Part III The effect of miR-133b on the pregnancy rate of polycystic ovary syndrome rats1.AIM To investigate the expression of miR-133b in the PCOS rat ovaries and its effect on the rat fertility and to explore the role of miR-133b in the pathogenesis of PCOS.2.Methods Letrozole was used to construct the animal model of polycystic ovary syndrome.PCOS rats were injected with miR-133b agonist and miR-133b antagonist,respectively.Analyze the peripheral blood hormone levels and the expression of TAGLN2 in PCOS rats.Normal control group,the polycystic ovary syndrome rat model group,the PCOS rat treated with miR-133b agonist group,and the PCOS rat treated with miR-133b antagonist group were caged with males.The pregnancy status of the rats in each group was recorded and pregnancy rate was calculated.3.Results 1).Under the optical microscope,the ovarian follicles of the rats with polycystic ovary syndrome were mostly cystic dilation,with reduced granulosa cell layers and destruction of the normal ovarian structure.In the rat ovary treated with miR-133b agonist group,most of the follicles were primary follicles,and the granulosa cell layer was orderly arranged,and the ovarian structure tended to be normal.In the rats ovary treated with miR-133b antagonist group,ovarian follicles showed more cystic expansion and granulosa cell layers decreased.2).The serum androgen level in PCOS rats was significantly higher than that of the other three groups,and the difference was significant(P<0.01).The serum androgen level in PCOS rats treated with miR-133b agonist was lower than that of in the PCOS rats.The serum luteinizing hormone,luteinizing hormone/follicle stimulating hormone levels in PCOS rats were significantly higher than those of in the control group.The serum luteinizing hormone,luteinizing hormone/follicle stimulating hormone levels in PCOS rats treated with miR-133b agonist were significantly lower than those of in the PCOS rats.3).The protein expression of TAGLN2 increased in PCOS rats and decreased in the PCOS rats treated with miR-133b agonist.Meanwhile,miR-133b antagonist could rescue the protein expression of TAGLN2 in PCOS rats.4)The pregnancy rate of rats in the control group was 100%,and the pregnancy rate of PCOS rats in the PCOS group was 33.33%.Interesting,the pregnancy rate of PCOS rats was up to 66.67% when treated with miR-133b agonist and the pregnancy rate of PCOS rats was 33.33% when treated with miR-133b antagonist.Conclusion 1)MiR-133b regulates the expression levels of TAGLN2 and caspase-3 in cumulus granulosa cells and promotes the proliferation of human cumulus granulosa cells;2)MiR-133b can improve PCOS symptoms and increase pregnancy rate,and its mechanism may be related to targeting TAGLN2.