摘要
目的探讨RNA干扰技术沉默人NOB1基因对人结肠癌RKO细胞增殖和凋亡的影响。方法设计靶向NOB1基因的小干扰RNA（siRNA），构建NOB1基因特异性的RNA干扰慢病毒载体，感染RKO细胞，并设置阴性对照组。实时定量PCR和Westemblot分别检测两组细胞NOB1mRNA及蛋白的表达情况；CellomicsArrayScanVTIHCS高内涵分析仪检测感染细胞的增殖和克隆形成能力；流式细胞术检测细胞周期及凋亡的变化：裸鼠成瘤实验检测0NOB1-siRNA对肿瘤的抑制作用。结果NOB1-shRNA慢病毒感染RKO细胞3d后．与阴性对照组比较．NOB1mRNA和蛋白的表达明显降低，克隆形成数减少，而细胞凋亡数增加（均P〈0．05）。裸鼠成瘤实验结果显示，NOB1．shRNA感染组裸鼠移植瘤体积为（405±102）mm3，小于阴性对照组的（870±165）mm3（P〈0．05）。结论通过RNA干扰方式沉默NOB1基因的表达．有望作为抑制结肠癌发展的有效手段。
Objective To investigate the effect of NOB1 gene on proliferation and apoptosis of human colon cancer cell line RKO by RNA interference. Methods Small interference RNA （siRNA） targeting NOB1 gene was cloned into lentivirus vector. Then the lentivirus particles expressing NOB1 short harpin RNA （shRNA） were infected into RKO cells. Real-time PCR and Western blot were performed to examine the expression of NOB1 in lentivirus infected cells. The Thermo Scientific Cellomics ArrayScan VTI HCS Reader was used to test the proliferation and colony-formation of RKO cells, and flow cytometry assay was performed to detect cell cycle and apoptosis. Xenograft tumor was established by injection of RKO cells into nude mice, then NOBI-shRNA was injected into the tumor and tumor volume was detected. Results Compared to negative controls, the expression levels of NOB1 mRNA and protein were both significantly down-regulated, the proliferation and colony-forming capacity of RKO cells were significantly inhibited, and cell apoptosis was increased after 3 days of NOB1-shRNA lentivirus infection （all P〈0.05）. The tumor volume was significantly smaller in NOB1- shRNA group than that in Scr-shRNA group [（405±102） mm3 vs. （870±165） mm3, P〈0.05]. Conclusion Silencing NOB1 gene by RNA interference may provide an inhibitive effect on human colon cancer development.
出处
《中华胃肠外科杂志》
CAS
2012年第11期1182-1186,共5页
Chinese Journal of Gastrointestinal Surgery
基金
浙江省医药卫生科技计划类基金2010KYA178
关键词
结肠肿瘤
RNA干扰
增殖
凋亡
Colonic neoplasms
RNA interference
Proliferation
Apoptosis
