miR-135a-5p通过Smad4基因调控多囊卵巢综合征卵巢颗粒细胞的增殖及凋亡

miR-135a-5p regulated proliferation and apoptosis of ovarian granulosa cells in polycystic ovary syndrome through Smad4 gene

目的探讨微小RNA-135a-5p(miR-135a-5p)通过调控SMAD家庭成员4(Smad4)表达而调控多囊卵巢综合征卵巢颗粒细胞的增殖及凋亡。方法体外培养人卵巢颗粒细胞KGN与人正常卵巢上皮细胞IOSE80,实时荧光定量聚合酶链反应(qRT-PCR)与蛋白免疫印迹法(Western blot)分别检测miR-135a-5p、Smad4的表达量;分别用anti-miR-con、anti-miR-135a-5p、anti-miR-135a-5p与si-con、anti-miR-135a-5p与si-Smad4转染至卵巢颗粒细胞;甲基噻唑基四唑法(MTT)检测细胞增殖;平板克隆实验检测细胞克隆形成能力;流式细胞术检测细胞凋亡率;双荧光素酶报告实验检测过表达miR-135a-5p对野生型和突变型Smad4荧光素酶活性的影响;Western blot检测细胞周期蛋白D1(Cyclin D1)、细胞周期依赖蛋白激酶2(CDK2)、B淋巴细胞瘤-2相关蛋白(Bax)、活化的含半胱氨酸的天冬氨酸蛋白水解酶3(Cleaved caspase-3)、B淋巴细胞瘤-2(Bcl-2)的表达量。结果与正常卵巢上皮细胞IOSE80比较,KGN细胞中miR-135a-5p的表达水平显著升高,Smad4 mRNA及蛋白表达量显著降低,差异均有统计学意义(P<0.05)。与anti-miR-con组比较,anti-miR-135a-5p组细胞活力显著降低,克隆形成数显著减少,细胞凋亡率显著升高,Cyclin D1、CDK2、Bcl-2蛋白水平显著降低,Bax、Cleaved caspase-3蛋白水平显著升高,差异均有统计学意义(P<0.05)。双荧光素酶报告实验证实miR-135a-5p能够抑制Smad4的3′-UTR区荧光素酶活性;与anti-miR-135a-5p+sicon组比较,anti-miR-135a-5p+si-Smad4组细胞活力显著升高,克隆形成数显著增多,细胞凋亡率显著降低,Cyclin D1、CDK2、Bcl-2蛋白水平显著升高,Bax、Cleaved caspase-3蛋白水平显著降低,差异均有统计学意义(P<0.05)。结论干扰miR-135a-5p可能通过靶向调控Smad4的表达从而抑制卵巢颗粒细胞增殖及促进细胞凋亡。

Objective To investigate whether miR-135 a-5 p could regulate the proliferation and apoptosis of ovarian granulosa cells in polycystic ovary syndrome by regulating SMAD family member 4(Smad4)expression.Methods Human ovarian granulosa cells KGN and human normal ovarian epithelial cells IOSE80 were cultured in vitro.The expressions of miR-135 a-5 p and Smad4 were detected by quantitative real-time PCR(qRT-PCR)and Western blot,respectively.Anti-miRcon,anti-miR-135 a-5 p,anti-miR-135 a-5 p and si-con,anti-miR-135 a-5 p and si-Smad4 were used to transfect into ovarian granulocytes.MTT was used to detect the cell proliferation.Plate cloning experiment was used to detect the cell clone formation ability.Flow cytometry was used to detect the apoptotic rate.The double luciferase reporter assay was used to detect the effect of miR-135 a-5 p overexpression on the activity of wild-type and mutant Smad4 luciferase.Western blot was used to detect the expression of Cyclin D1,cyclin dependent kinase 2(CDK2),Bcl-2-associated X protein(Bax),Cleaved caspase-3,and B-cell lymphoma-2(Bcl-2).Results Compared with IOSE80,the expression level of miR-135 a-5 p in KGN cells was significantly increased(P<0.05),and the expressions of Smad4 mRNA and protein levels were significantly reduced(P<0.05).Compared with the anti-miR-con group,the cell viability of the anti-miR-135 a-5 p group was significantly reduced(P<0.05),the number of colony formation was significantly reduced(P<0.05),the apoptosis rate was significantly increased(P<0.05),the levels of Cyclin D1,CDK2,and Bcl-2 protein were significantly reduced(P<0.05),and the levels of Bax,Cleaved caspase-3 protein were significantly increased(P<0.05).Double luciferase reporter assay was used to detect the effect of overexpressing miR-135 a-5 p on wild-type and mutant Smad4 luciferase activity.Compared with the anti-miR-135 a-5 p+si-con group,the cell viability of the anti-miR-135 a-5 p+si-Smad4 group was significantly increased(P<0.05),the number of colony formation was significantly increased(P<0.05),apoptosis rate was significantly reduced(P<0.05),the levels of Cyclin D1,CDK2,and Bcl-2 protein were significantly increased(P<0.05),and the levels of Bax,Cleaved caspase-3 protein were significantly reduced(P<0.05).Conclusion Interference with miR-135 a-5 p might inhibit the proliferation of ovarian granulosa cells and promote apoptosis by regulating the expression of Smad4.