摘要
为了研究整合素连接激酶(integrin-linked kinase,ILK)基因沉默对膀胱癌BIU-87细胞系增殖产生的影响,应用RNA干扰(RNA interference,RNAi)技术,合成了4条针对ILK的miRNA干扰载体pcDNATM6.2-GW/EmGFP-ILK-miR(miR-1、miR-2、miR-3、miR-4)作为实验组,另设1条为阴性对照组,分别转染膀胱癌BIU-87细胞系.经杀稻瘟菌素持续压力筛选和有限稀释法培养获得稳定转染细胞株.采用RT-PCR和Western-blot检测各组对ILK基因的抑制作用,实验组miRNA对BIU-87细胞ILK mRNA和蛋白的表达均有明显的抑制作用,以miR-3组抑制效应最强.通过流式细胞术检测发现,miR-3组细胞周期G0/G1期细胞所占比例较未转染组明显增加,而S期则明显减少(P<0.05).四甲基偶氮唑盐(MTT)比色法检测发现,转染组细胞在体外的增殖能力较对照组明显下降(P<0.05).证明RNA干扰技术能有效抑制靶基因ILK的表达,进而抑制了膀胱癌细胞系BIU-87的增殖.
To study the effect of RNAi on the expression of ILK mRNA and protein and its influence on the proliferation in bladder cancer BIU-87 cell line,RNA interference（RNAi） was applied and four specific miRNA RNAi vectors pcDNATM6.2-GW/EmGFP-ILK-miR（miR-1,miR-2,miR-3,miR-4）targeting human ILK and a negative control group were synthesized and transfected into the BIU-87 cells by liposome.The stable expression cell strain were obtained by continuing pressure screening of blasticidin and limiting dilution assay cultivation.The suppression effect of each group targeting ILK was identified by RT-PCR and Western-blot.The experimental group miRNA significantly inhibited the expression of ILK mRNA and protein in BIU-87 cells,and miR-3 group showed the strongest inhibitory effect.Detected by flow cytometry,in miR-3 transfected group,the number of cells in cell cycle G0 / G1 phase cells was significantly increased compared with non-transfected,while that of the S phase was significantly decreased（P 〈0.05）.Methylthiazolyl tetrazolium（MTT） colorimetric assay showed that the proliferation in transfected group decreased significantly compared with the control group（P〈 0.05）.It was demonstrated that RNA interference can effectively inhibit the expression of target gene ILK,thereby inhibit proliferation of bladder cancer cell line BIU-87.
出处
《生命科学研究》
CAS
CSCD
2010年第6期528-533,共6页
Life Science Research
基金
国家自然科学基金资助项目(30972999)
重庆市自然科学基金资助项目(CSTC2005BB5033)
