慢病毒载体介导RNA干扰抑制大肠癌细胞HT29的酪氨酸激酶受体RON基因的表达  

Lentiviral vector mediated RNA interference inhibits RON gene expression in the human colorectal carcinoma HT29 cells

作　　者：毛联钢[1] 李克强[1] 卓文莹[2] 戴晓宇[2] 余永明[2] 乐东海[1] 冯伟云[1] 

机构地区：[1]宁波市第二医院肿瘤分子生物学重点实验室,浙江宁波315000 [2]宁波市第二医院肛肠外科,浙江宁波315000

出　　处：《上海医学》2012年第11期935-939,I0003,共6页Shanghai Medical Journal

基　　金：宁波市自然科学基金资助项目(2008A610082)

摘　　要：目的研究慢病毒介导的RNA干扰(RNAi)对大肠癌细胞RON基因的干扰效率,构建RON基因稳定沉默的大肠癌细胞株。方法应用实时聚合酶链反应(PCR)检测大肠癌细胞DLD-1、HCT116、LOVO、RKO、SW480、HT29的RONmRNA表达情况,筛选出表达丰度能满足RNAi的大肠癌靶细胞HT29。根据人RON基因序列,构建4种慢病毒载体质粒pGCSIL/RON-短发夹RNA(shRNA),转染包装细胞293T,获得4种重组慢病毒pGCSIL/RON-shRNA,分别感染HT29细胞。应用实时PCR检测各组HT29细胞RONmRNA表达情况,筛选出RON干扰效率最高的一种shRNA慢病毒载体质粒,构建RON基因稳定沉默的大肠癌细胞株HT29/RON-shRNA。结果 DLD-1、HCT116、RKO、SW480、HT29细胞RON表达丰度能满足RNAi的需要,选择HT29细胞作为RNAi的靶细胞。阴性对照病毒感染的细胞样品相对正常未感染病毒的细胞目的基因的相对表达水平为(94±5)%,RONshRNA-1靶点病毒感染的细胞为(86±5)%,RONshRNA-2靶点病毒感染的细胞为(67±6)%,RONshRNA-3靶点病毒感染的细胞为(63±6)%,RONshRNA-4靶点病毒感染的细胞为(30±8)%,RONshRNA-4靶点病毒感染的细胞的表达水平显著低于其他细胞(P值均<0.01),其对目的基因的沉默效果达到(70±8)%。结论慢病毒介导RNAi能显著抑制大肠癌细胞HT29的RON基因的表达,慢病毒载体介导的RNAi是一种高效的"基因沉默"的手段。Objective To study the RNA interference （RNAi） with RON genes in the human colorectal carcinoma cells through lentiviral vector, and to establish the human colorectal carcinoma cell line in which the RON mRNA expression was stably suppressed. Methods The RON mRNA expression in DLD-1, HCT116, LOVO, RKO, SW480 and HT29 cells was detected with real-time polymerase chain reaction （RT-PCR）. Then the human colorectal carcinoma cell line HT29 in which the RON expression was conformed to the RNAi was screened. According to human RON gene sequencing, 4 kinds of recombinant lentiviral vector pGCSIL/RON-short hairpin RNA （shRNA） were constructed and transferred into packaging cell line 293T, then 4 kinds of recombinant lentivirus were obtained and HT29 cells were infected in vitro. The expression of RON mRNA in infected HT29 cells was detected with RT-PCR. Recombinant lentiviral Vector pGCSIL/RON-shRNA, which interfered in the expression of RON mRNA in HT29 cells most effectively, were screened out. The human colorectal carcinoma cell line HT29/ RON-shRNA in which the RON genes were stably knocked down was established. Results The RON expression in the human colorectal carcinoma cell line DLD-1, HOT116, RKO, SW480 and HT29 were conformed to the RNAi. HT29 cells were selected as the target cells of RNAi. Compared to control cells, the expression of target genes in negative control of virus infected cells, RON targeting shRNA-1 virus infected cells, RON targeting shRNA-2 virus infected cells, RON targeting shRNA-3 virus infected cells, RON targeting shRNA-4 virus infected cells were （94±5）%, （86±5）%, （67±6）%, （63±6）% and （30±8）%, respectively. The expression of RON targeting shRNA-4 virus infected cells was significantly lower than the other cells （all P〈0.01）. The RON mRNA expression in HT29/RON-shRNA cells decreased by （70 ±8） %. Conclusion Lentiviral vector-induced RNAi can significantly suppress the RON mRNA expression in HT29 cells, and it is an effective ＂gene silence＂

关 键 词：RON HT29细胞 RNA干扰 慢病毒载体 

分 类 号：R735.34[医药卫生—肿瘤]