微小RNA-873-5p通过靶向作用于PYGO2基因对膀胱癌细胞生长的影响  

Effect of MicroRNA-873-5p on the growth of bladder cancer cells by targeting PYGO2 gene

作　　者：王勇[1] 郭永连[1] 陈琳[1] 李国灏[1] 余家俊[1] 程薇[2] Wang Yong;Guo Yonglian;Chen Lin;Li Guohao;Yu Jiajun;Cheng Wei(Department of Urology,The Central Hospital of Wuhan,Tongji Medical College, Huazhong University of Science and Technology,Wuhan Hubei 430014,China;Department of Otolaryngology,Liyuan Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan Hubei 430077,China)

机构地区：[1]华中科技大学同济医学院附属武汉中心医院泌尿外科,湖北武汉430014 [2]华中科技大学同济医学院附属梨园医院耳鼻喉科,湖北武汉430077

出　　处：《遵义医学院学报》2017年第6期636-640,645,共6页Journal of Zunyi Medical University

基　　金：湖北省自然科学基金资助项目(NO:2017CFB176)

摘　　要：目的探讨微小-873-5p(miR-873-5p)转染对PYGO2基因表达的调控作用及对膀胱癌细胞5637生长的影响。方法根据处理不同,膀胱癌细胞分为对照组(转染miR-NC)和实验组(转染miR-873-5p)。qRT-PCR检测转染后两组细胞中miR-873-5p表达量。生物信息学预测PYGO2为miR-873-5p可能的靶基因。qRT-PCR检测PYGO2mRNA的表达。构建PYGO2基因的3’UTR野生型及突变型序列双荧光素酶报告基因载体并进行荧光素酶活性检测。Western blot检测PYGO2及下游靶蛋白的表达。流式细胞术分析细胞周期分布,MTS法和集落形成实验分析细胞活力和增殖能力。结果实验组中miR-873-5p表达量显著上升(P<0.01)。双荧光素酶报告基因系统结果显示PYGO2是miR-873-5p的靶基因。与对照组相比,实验组细胞中PYGO2 mRNA水平下调58.48%(P<0.01)。PYGO2及下游靶蛋白表达明显下调。转染miR-873-5p后,细胞周期被阻滞在G0/G1期(P<0.01),实验组细胞的活力和增殖能力均明显降低(P<0.05)。结论 miR-873-5p通过干扰膀胱癌细胞5637中PYGO2基因的表达,阻滞细胞周期进展,抑制细胞的增殖,可能成为膀胱癌基因治疗的靶标分子。Objective To investigate the effect of miR-873-5p transfection on the expression of PYGO2gene and the effect on the growth of bladder cancer cell line5637.Methods According to the different treatments,bladder cancer cells were divided into control group(transfected with miR-NC)and experimental group(transfected with miR-873-5p).qRT-PCR was used to detect the expression of miR-873-5p in the two groups after transfection.Bioinformatics predicts that PYGO2is a possible target gene for miR-873-5p.The expression of PYGO2mRNA was detected by qRT-PCR.3’UTR wild type and mutant sequence double luciferase reporter gene vector of PYGO2gene were constructed and luciferase activity was measured.Western blot was used to detect the expression of PYGO2and downstream target protein.Flow cytometry was conducted to analyze cell cycle distribution;MTS assay and colony formation assay were performed to analyze cell viability and proliferative capacity.Results The expression of miR-873-5p in the experimental group was significantly increased(P<001).Double luciferase reporter gene system results show that PYGO2is a target gene for miR-873-5p.Compared with the control group,the level of PYGO2mRNA in the experimental group was down-regulated by5848%(P<0.01).The expression of PYGO2and downstream target proteins were down-regulated.After transfection of miR-873-5p,the cell cycle was arrested at G0/G1phase(P<0.01).The viability and proliferation ability of the experimental group were significantly decreased(P<0.05).Conclusion miR-873-5p can block cell cycle progression and inhibit cell proliferation of bladder cancer by interfering with the expression of PYGO2gene,and may be a target molecule for gene therapy of bladder cancer.

关 键 词：微小RNA PYGO2 膀胱癌 C-Myc 细胞周期蛋白D1 CDK4 

分 类 号：R737.14[医药卫生—肿瘤]