微小RNA-8085在膀胱癌组织中的表达及对膀胱癌J82细胞侵袭和增殖的影响

Expression of microRNA-8085 in bladder cancer and its effect on invasion and proliferation of bladder cancer cell line J82

目的分析微小RNA-8085(miR-8085)在膀胱癌组织中的表达及其对膀胱癌J82细胞侵袭和增殖的影响,并探究其分子机制。方法实时定量PCR(qPCR)检测32例膀胱癌和癌旁组织中miR-8085的表达水平。以携带miR-8085的慢病毒或阴性对照慢病毒感染J82细胞,命名为实验组和对照组,qPCR检测转染效率。Transwell侵袭实验和四甲基偶氮唑蓝(MTT)法分别检测感染后J82细胞的侵袭能力和增殖能力。生物信息学方法预测miR-8085可能的靶基因。双荧光素酶报告基因实验验证miR-8085与靶基因mRNA的结合。qPCR和Westernblot检测感染后J82细胞中靶基因在mRNA和蛋白水平的表达量。结果膀胱癌组织和癌旁组织中miR-8085的表达量分别为2.50±0.78和7.35±0.92(P<0.01)。与对照组相比,实验组J82细胞中miR-8085的表达量显著增加(P<0.01)。对照组和实验组中J82穿膜细胞数分别为82.63±7.60和35.45±11.27,实验组J82细胞的侵袭能力显著被抑制(P<0.05)。实验组J82细胞的增殖能力从第3天开始显著被抑制(P<0.05)。生物信息学方法显示,miR-8085可能的靶基因是拓扑异构酶Ⅱα(TOP2A)。双荧光素酶报告基因实验显示,miR-8085可与TOP2AmRNA3′非翻译区靶向结合。实验组J82细胞株中TOP2A基因在mRNA和蛋白水平的表达量均显著减少(P<0.01)。结论miR-8085在膀胱癌中表达明显降低,过表达miR-8085可通过干扰TOP2A基因的表达,抑制膀胱癌J82细胞的侵袭和增殖能力,可能成为膀胱癌治疗的潜在靶点。

Objective To analyze the expression of microRNA-8085(miR-8085)in bladder cancer tissues and its effect on invasion and proliferation of bladder cancer J82 cells,and to explore its molecular mechanism.Methods Real-time quantitative PCR(qPCR)was used to detect the expression level of miR-8085 in 32 cases of bladder cancer and adjacent tissues.J82 cells were infected with a lentivirus carrying a miR-8085(experimental group)or a negative control lentivirus(control group).The qPCR was used to detect the transfection efficiency.Transwell invasion assay and MTT assay were used to detect the invasive and proliferative capacities of J82 cells after infection.Bioinformatics methods were used to predict possible target genes for miR-8085.The dual luciferase reporter gene assay was used to verify the binding of miR-8085 to the target gene mRNA.The expression levels of target genes at mRNA and protein levels in J82 cells after infection were detected by qPCR and Western blot.Results The expression levels of miR-8085 were 2.50±0.78 and 7.35±0.92 in bladder cancer tissues and adjacent tissues,respectively(P<0.01).The expression of miR-8085 was significantly higher in experimental group than in control group(P<0.01).The number of transmembrane cells in control group and experimental group was 82.63±7.60 and 35.45±11.27,respectively(P<0.05).The invasive ability of J82 cells in experimental group was signi-ficantly inhibited compared with control group(P<0.05).Compared with control group,the proliferative capacity in experimental group J82 cells was significantly inhibited from the third day after inoculation of cells(P<0.05).Bioinformatics methods showed that the possible target gene for miR-8085 was topoisomeraseⅡα(TOP2A).Dual luciferase reporter assays showed that miR-8085 can bind to the 3′untranslated region of TOP2A mRNA.The expression levels of TOP2A gene in J82 cells were significantly decreased in experimental group at mRNA and protein levels(P<0.01).Conclusion The expression of miR-8085 is significantly decreased in bladder cancer.Overexpression of miR-8085 could inhibit the invasion and proliferation of bladder cancer J82 cells by interfering the expression of TOP2A gene,which may be a potential target for bladder cancer treatment.