论文部分内容阅读
目的:探讨微RNA-34b(microRNA-34b,miR-34b)对人膀胱癌BIU-87细胞增殖的影响及其可能的作用机制。方法:应用实时荧光定量PCR检测膀胱癌BIU-87细胞、人膀胱上皮永生化细胞SV-HUC-1和人肾小管上皮细胞HK-2中miR-34b的表达水平。将miR-34b mimic转染至BIU-87细胞后,应用MTT法和克隆形成实验检测BIU-87细胞的增殖和克隆形成情况。miR-34b mimic与泛素特异肽酶22(ubiquitin-specific peptidase 22,USP22)-野生型(wide type,WT)或USP22-突变型(mutation type,Mut)重组载体共转染后,应用萤光素酶报告系统检测miR-34b是否与USP22基因3’-非翻译区(3’-untranslated region,3’-UTR)结合。应用实时荧光定量PCR和蛋白质印迹法检测miR-34b mimic转染后,BIU-87细胞中USP22 mRNA和蛋白的表达水平。结果:膀胱癌BIU-87细胞中miR-34b的表达水平低于肾小管上皮细胞HK-2和膀胱上皮永生化细胞SV-HUC-1(P值均<0.05)。miR-34b mimic转染后,BIU-87细胞的增殖率和克隆形成率低于空白对照组(BIU-87细胞未进行任何转染)和阴性对照组[BIU-87细胞转染miRNA mimic negative control(miRNA mimic NC)](P值均<0.05)。萤光素酶报告系统检测结果显示,miR-34b能特异性地与USP22基因3’-UTR结合,miR-34b mimic与重组载体USP22-WT共转染组细胞的萤光素酶活性下降(P<0.05)。miR-34b mimic转染组BIU-87细胞中USP22蛋白的表达水平低于空白对照组和阴性对照组(P值均<0.05)。结论:miR-34b可抑制膀胱癌BIU-87细胞的增殖,这一作用可能与USP22的表达有关。
Objective: To investigate the effect of microRNA-34b (miR-34b) on the proliferation of human bladder cancer BIU-87 cells and its possible mechanism. Methods: The expression of miR-34b in bladder cancer BIU-87 cells, human bladder epithelial immortalized cells SV-HUC-1 and human renal tubular epithelial cells HK-2 were detected by real-time fluorescence quantitative PCR. After transfection of miR-34b mimic into BIU-87 cells, the proliferation and colony formation of BIU-87 cells were detected by MTT assay and clonogenic assay. After co-transfection of miR-34b mimic with ubiquitin-specific peptidase 22 (USP22) -wild type (WT) or USP22-mutant type (Mut) recombinant vector, The enzyme reporter system detects whether miR-34b binds to the USP22 3’-untranslated region (3’-UTR). Real-time quantitative PCR and Western blotting were used to detect the expression of USP22 mRNA and protein in BIU-87 cells after miR-34b mimic transfection. Results: The expression level of miR-34b in bladder cancer BIU-87 cells was lower than that in HK-2 renal tubular epithelial cells and SV-HUC-1 in bladder epithelial immortalized cells (all P <0.05). After transfected with miR-34b mimic, the proliferation rate and clonogenic rate of BIU-87 cells were lower than that of the blank control group (no transfection of BIU-87 cells) and the negative control group [miRNA mimic negative control (miRNA mimic NC)] (all P <0.05). The results of luciferase reporter assay showed that miR-34b could specifically bind to 3’-UTR of USP22 gene and the luciferase activity of miR-34b mimic and co-transfection group of recombinant vector USP22-WT decreased (P <0.05). The expression level of USP22 protein in BIU-87 cells transfected with miR-34b mimic group was lower than that in blank control group and negative control group (all P <0.05). Conclusion: miR-34b can inhibit the proliferation of bladder cancer BIU-87 cells, which may be related to the expression of USP22.