miR-140-5p靶向调控HAND2影响TGF-β1诱导肾小管上皮细胞生长

MiR-140-5p targets HAND2 and affects TGF-β1 induced renal tubular epithelial cell

目的探讨miR-140-5p调控心脏神经脊衍生物表达转录因子2(HAND2)对转化生长因子β1(TGF-β1)诱导肾小管上皮细胞生长的影响。方法实验设置TGF-β1组、对照(NC)组、miR-NC组、miR-140-5p组、anti-miRNC组、anti-miR-140-5p组、miR-NC+TGF-β1组、miR-140-5p+TGF-β1组、si-NC+TGF-β1组、si-HAND2+TGF-β1组、miR-140-5p+pcDNA-NC+TGF-β1组、miR-140-5p+pcDNA-HAND2+TGF-β1组。实时荧光定量PCR(RT-qPCR)检测miR-140-5p和HAND2 mRNA表达水平;蛋白质印迹(Western blot)法检测HAND2、细胞周期蛋白D1(CyclinD1)、裂解的半胱氨酸天冬氨酸蛋白酶-3(Cleaved-caspase-3)、磷酸化磷脂酰肌醇3激酶(p-PI3K)、磷酸化蛋白激酶B(p-AKT)蛋白表达;四甲基偶氮唑盐比色法(MTT)检测细胞增殖率;流式细胞术检测细胞凋亡;双荧光素酶报告实验验证miR-140-5p和HAND2的靶向关系。结果TGF-β1诱导的肾小管上皮细胞中miR-140-5p低表达,HAND2高表达。高表达miR-140-5p或低表达HAND2,TGF-β1诱导的肾小管上皮细胞中CyclinD1表达水平升高,Cleaved-caspase-3表达水平降低,细胞增殖率升高,细胞凋亡率降低(P<0.05)。miR-140-5p靶向调控HAND2,高表达HAND2可以逆转miR-140-5p对TGF-β1处理的肾小管上皮细胞增殖和凋亡的影响。高表达miR-140-5p,p-PI3K、p-AKT表达水平升高;而高表达HAND2逆转了miR-140-5p对PI3K/AKT信号通路的促进作用。结论过表达miR-140-5p通过靶向下调HAND2激活PI3K/AKT信号通路促进肾小管上皮细胞增殖,抑制响TGF-β1诱导的肾小管上皮细胞凋亡。

ObjectiveTo investigate the effect of mi R-140-5 p regulating heart and neural crest derivatives-ex-pressed transcript 2(HAND2)on transforming growth factor beta1(TGF-β1)induced renal tubular epithelial cell growth.MethodsThe experimental set TGF-β1 group,control group(NC),mi R-NC group,mi R-140-5 p group,anti-mi R-NCgroup,anti-mi R-140-5 p group,mi R-NC+TGF-β1 group,mi R-140-5 p+TGF-β1 group,si-NC+TGF-β1 group,si-HAND2+TGF-β1 group,mi R-140-5 p+pc DNA-NC+TGF-β1 group,mi R-140-5 p+pc DNA-HAND2+TGF-β1 group.Real-time quanti-tative PCR(RT-q PCR)was used to detect mi R-140-5 p and HAND2 m RNA expressions;Western blot was used to detectHAND2,Cyclin D1(Cyclin D1),cleaved cysteine aspartic protease-3(Cleaved-caspase-3),phosphorylated phosphati-dylinositol 3 kinase(p-PI3 K),phosphorylated protein kinase B(p-AKT)protein expression;Tetramethylazozolium colorim-etry(MTT)was used to detect cell proliferation rate;flow cytometry was used to detect apoptosis;dual luciferase report ex-periments were performed to verify the targeting relationship between mi R-140-5 p and HAND2.Results Low expression ofmi R-140-5 p and high expression of HAND2 in TGF-β1-induced renal tubular epithelial cells.High expression of mi R-140-5 por low expression of HAND2,Cyclin D1 expression was increased in renal tubular epithelial cells induced by TGF-β1,Cleaved-caspase-3 expression was decreased,cell proliferation rate was increased,and apoptosis rate was decreased(P<0.05).mi R-140-5 p targets HAND2,and overexpression of HAND2 can reverse the effects of mi R-140-5 p on the prolifera-tion and apoptosis of renal tubular epithelial cells treated with TGF-β1.High expression of mi R-140-5 p,p-PI3 K,p-AKT expressions were increased;and high expression of HAND2 reversed the mi R-140-5 p on promotion of PI3 K/AKT signalingpathway.ConclusionOverexpression of mi R-140-5 p can promote the proliferation of renal tubular epithelial cells by target-ing down-regulated HAND2 to activate the PI3 K/AKT signaling pathway,and inhibit renal tubular epithelial cell apoptosisinduced by TGF-β1.