过表达miRNA-217由P13/AKT信号通路促进膀胱癌T24细胞增殖作用

Effects of overexpression of mRNA217 in promoting the proliferation of bladder cancer T24 cells through the P13/AKT signaling pathway

目的研究miRNA-217是否通过P13/AKT信号通路上调E2F3促进膀胱癌T24细胞增殖作用。方法收集2017年12月至2018年4月收治的64例膀胱癌患者临床组织。QRT-PCR分别肿瘤组织和癌旁组织中miRNA-217的表达;Western blot检测肿瘤组织和癌旁组织中PI3K、P-AKT、E2F3表达;转染过表达载体miRNA-217上调T24细胞中miRNA-217的表达作为RSK4-miRNA-217组,空白载体microRNA-NC转染T24细胞为RSK4-NC组,以未转染的细胞为空白对照组。qRT-PCR检测3组细胞miRNA-217的表达;CCK-8及平板克隆形成实验、裸鼠皮下荷瘤模型检测3组细胞的增殖及克隆形成能力及成瘤能力;荧光素梅实验观察miRNA-217和PI3K的关系;Western blot检测3组细胞中PI3K、P-AKT、E2F3的表达。结果肿瘤组织中miRNA-217、PI3K、P-AKT和E2F3的表达高于癌旁组织;与空白对照组和RSK4-NC组比较,RSK4-miRNA-217组细胞OD450、细胞克隆数明显增加,移植瘤体积明显增大(P<0.05),空白对照组和RSK4-NC组间差异无统计学意义(P>0.05);经预测发现miRNA-217和PI3K有互补结合序列,双荧光素酶报告实验证实两者的靶向结合关系;与空白对照组和RSK4-NC组比较,RSK4-miRNA-217组细胞PI3K、p-AKT、E2F3蛋白表达明显升高(P<0.05),在空白对照组和RSK4-NC组细胞中差异无统计学意义(P>0.05)。结论miRNA-217通过P13/AKT信号通路上调E2F3从而促进膀胱癌T24细胞增殖作用。

Objective To investigate whether miRNA217 can promote the proliferation of bladder cancer T24 cells through upregulating E2F3 via the P13/AKT signaling pathway.Methods A total of 64 tissue specimens of patients with bladder cancer who were admitted and treated in our hospital from December 2017 to April 2018 were collected.The expression levels of miRNA-217 in tumor tissues and adjacent tissues were detected by qRT-PCR.The expression levels of PI3K,P-AKT,and E2F3 in tumor tissues and paracancer tissues were detected by Western Blot.The T24 cells which were upregulated the expressions of miRNA-217 through transfecting the overexpression vector-miRNA-217 were enrolled as RSK4 miRNA-217 group.The T24 cells which were transfected by blank vector microRNA-NC were enrolled as RSK4-NC group.The cells without transfection were enrolled as blank control group.The expression levels of miRNA-217 in the three groups were detected by qRT-PCR.The cells proliferation was detected by CCK8 and the cell growth condition was detected by plate clone formation assay.The proliferation,clonality and tumor capacity of the cells in the three groups were detected by nude mouse subcutaneous tumor bearing model.Moreover the correlation between miRNA-217 and PI3K was observed in luciferin plum experiment.The expression levels of PI3K,P AKT and E2F3 were detected by Western Blot.Results The expression levels of miRNA-217,PI3K,PAKT and E2F3 in tumor tissues were significantly higher than those in para-carcinoma tissues.The OD450,number of cell clones and the volume of transplantation tumor in the RSK4 miRNA-217 were significantly increased,as compared with those in blank control group and RSK4-NC group(P<0.05),however,there were no significantly differences between blank control group and the RSK4-NC group(P>0.05).The website of www.microRNA.org showed that miRNA-217 and PI3K had complementary binding sequences.The targeting binding correlation was confirmed by dual luciferase reporting experiment.The protein expression levels of PI3K,pAKT,E2F3 in RSK4 miRNA-217 cells group were significantly increased,as compared with those in blank control group and the RSK4-NC group(P<0.05),however,there were no significantly differences between blank control group and the RSK4-NC group(P>0.05).Conclusion The miRNA-217 can upregulate E2F3 via P13/AKT signaling pathway so as to promote the proliferation of bladder cancer T24 cells.