ZONAB和E-cadherin在膀胱癌细胞系中的表达及RNA干扰ZONAB表达对癌细胞侵袭力的影响

摘要

目的:探讨慢病毒介导ZONAB基因的RNA干扰对膀胱癌侵袭力的影响.方法:用Realtime PCR法分别检测人类尿路上皮永生细胞系sv-huc-1和膀胱尿路上皮癌细胞系UM-UC-3、T24、5637中ZONAB和E-cadherin的mRNA表达水平,用Western blot法分别检测各细胞系中ZONAB蛋白表达水平,设计、合成干扰靶点,构建纯化重组慢病毒,感染人膀胱癌UM-UC-3细胞,根据ZONAB基因的抑制率,筛选最有效的ZONAB基因RNAi靶序列组,验证ZONAB基因的沉默效果,用Transwell侵袭试验检测UM-UC-3细胞体外侵袭力的改变.结果:Realtime PCR法和Western blot法测得UM-UC-3、T24、5637中ZONAB的表达水平高于sv-huc-1,均具有显著性差异(P<0.05);而Realtime PCR法测得UM-UC-3、T24、5637中 E-cadherin的mRNA表达水平低于sv-huc-1(P<0.05).成功构建ZONAB基因RNA干扰慢病毒载体并获得相应的慢病毒,与对照组相比RNA干扰组UM-UC-3细胞ZONAB基因mRNA水平的抑制率为71.4%(P<0.05),蛋白水平抑制率为76.8%,其穿过人工基底膜的平均细胞数为13.1±2.8/HP,明显少于阴性对照组和空白对照组(P<0.05).结论:ZONAB在膀胱癌细胞系中表达上调,慢病毒介导ZONAB基因的RNA干扰能够抑制UM-UC-3细胞侵袭.%Objective:To investigate the effect of ZONAB RNA interference on invasiveness of bladder cancer mediated by lentivirus vector.Methods:Realtime PCR was used to analyze the mRNA expression levels of ZONAB and E-cadherin in human urothelium immortalized cell line sv-huc-1 and bladder urothelial carcinoma cell lines UM-UC-3,T24,5637.The expression level of ZONAB protein in each cell line was analyzed by Western blot.Interfering targets were designed and synthesized.Recombinant lentivirus lentivirus was packaged and purified.Human bladder cancer UM-UC-3 cells were infected by lentivirus.According to ZONAB gene inhibition rate,the most effective ZONAB gene RNAi target sequence group was screened.ZONAB gene silencing effect was verified.In vitro invasion capability of UM-UC-3 cells was detected by Transwell invasion test.Results:The expression level of ZONAB gene in UM-UC-3,T24,5637 was higher than that in sv-huc-1 analyzed by Realtime PCR and Western blot,respectively(P<0.05).The expression of E-cadherin mRNA in UM-UC-3,T24,5637 was lower than that in sv-huc-1 detected by Realtime PCR(P<0.05).ZONAB gene RNA interference lentiviral vector was constructed and corresponding lentivirus was obtained.Compared with the control group,ZONAB gene mRNA inhibition rate in RNA interfering group of UM-UC-3 cells was 71.4%(P<0.05),and the protein inhibition rate was 76.8%(P<0.05).The average cell number migrated through the artificial basement membrane in RNA interfering group was 13.1±2.8/HP,which was significantly lower than that in negative control group and blank control group(P<0.05).Conclusion:The expression of ZONAB is up-regulated in bladder cancer cell lines.Lentivirus-mediated RNA interference of ZONAB gene can inhibit the invasion of UM-UC-3 cells.