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.
出处
《现代肿瘤医学》
CAS
2017年第18期2878-2882,共5页
Journal of Modern Oncology
基金
国家自然科学基金青年基金资助项目(编号:81501292)
辽宁省自然科学基金优秀人才培育项目(编号:2014021052)
