miR-142-3p对膀胱癌细胞增殖和有氧糖酵解的影响及作用机制

Effects of miR-142-3p on proliferation and aerobic glycolysis of bladder cancer cells and its mechanism

目的探究微小RNA(miR)-142-3p对膀胱癌(BC)细胞增殖与有氧糖酵解的影响及作用机制。方法qPCR检测比较人BC细胞系(SW780、UMUC3、T24、BIU87)和人输尿管上皮细胞(SV-HUC-1)中miR-142-3p表达水平,选取低表达的BC细胞进行miR-142-3p过表达质粒转染;分别采用平板细胞克隆实验、CCK8-法、流式细胞术、2-NBDG葡萄糖摄取实验、糖代谢检测试剂盒检测miR-142-3p表达对BC细胞增殖、活性、周期、有氧糖酵解的影响。双荧光素酶和Western blot法检测miR-142-3p与细胞周期蛋白依赖性激酶(CDK)4、乳酸脱氢酶A(LDHA)的靶向性及作用关系。过表达miR-142-3p的BC细胞分别转染CDK4或LDHA表达质粒,采用CCK8-法、2-NBDG葡萄糖摄取实验、糖代谢检测试剂盒测定转染后的BC细胞活性和有氧糖酵解情况。结果miR-142-3p在T24、UMUC3细胞中显著低表达(均P<0.01)。T24、UMUC3细胞转染过表达miR-142-3p质粒后,克隆能力及活性显著下降(均P<0.05),细胞周期阻滞于G0/G1期(均P<0.01),2-NBDG荧光强度显著减弱(均P<0.01),葡萄糖消耗量和乳酸生成量显著减少(均P<0.01)。miR-142-3p靶向抑制CDK4和LDHA蛋白表达(均P<0.01)。CDK4或LDHA表达显著逆转了miR-142-3p过表达对T24细胞的活性和有氧糖酵解的抑制作用(均P<0.05)。结论miR-142-3p通过靶向CDK4和LDHA抑制BC细胞增殖和有氧糖酵解。

Objective To investigate the effects of microRNA(miR)-142-3 on proliferation and aerobic glycolysis of bladder cancer(BC)cells and its mechanism.Methods Expression of miR-142-3p in BC cell lines(SW780,UMUC3,T24,BIU87)and human ureter SV-HUC-1 cells were detected by qPCR,then BC cell line with low miR-142-3p expression were transfected with miR-142-3p overexpression plasmid.Plate cloning test,CCK-8 assay,flow cytometry,2-NBDG glucose uptake assay and glucose metabolism detection kit were used to detect the effects of expression of miR-142-3p on proliferation,viability,cell cycle,aerobic glycolysis of BC cells.Luciferase report assay and Western blot were used to validate the relationship between cyclin-dependent kinase(CDK)4,lactate dehydrogenase A(LDHA)and miR-142-3p.The miR-142-3p-overexpressing BC cells were transfected with CDK4 or LDHA overexpression plasmid.CCK-8 assay,2-NBDG glucose uptake assay,and glucose metabolism detection kit were used to detect activities and aerobic glycolysis of transfected BC cells.Results Compared to other BC cell lines the expression of miR-142-3p in T24 and UMUC3 cells were significant lower(P<0.01).T24 and UMUC3 cells were transfected with miR-142-3p overexpressing plasmid.After transfection the cell cloning ability and viability were significantly decreased(all P<0.05),cell cycle were arrested in G0/G1phase(both P<0.01),2-NBDG fluorescence intensity were significantly weakened(both P<0.01),and glucose consumption and lactate production were significantly reduced(all P<0.01).The expression of CDK4 and LDHA proteins in miR-142-3poverexpressing BC cells was significantly inhibited(both P<0.01);the overexpressing of CDK4 or LDHA reversed the inhibition effects of miR-142-3p on T24 cell viability and aerobic glycolysis(P<0.05).Conclusion The miR-142-3p suppresses BC cell proliferation and aerobic glycolysis through targeting CDK4 and LDHA.