RNA干扰靶向抑制自噬调控基因Beclin 1对结肠癌细胞株HCT116自噬活性及增殖/凋亡的影响  

Inhibitory effects of Beclin 1 gene expression on the activities of autophagy and proliferation/apoptosis of colon cancer cell line HCT116 by RNA interference

作　　者：吴淑华[1] 何双[1] 胡金龙[1] 温菲菲 孙晨博 田东[1] 

机构地区：[1]滨州医学院附属医院病理科,滨州256603

出　　处：《临床与实验病理学杂志》2014年第6期599-604,共6页Chinese Journal of Clinical and Experimental Pathology

基　　金：山东省科技发展计划项目(2010GSF10259)

摘　　要：目的应用RNA干扰(RNA interference,RNAi)技术抑制自噬调控基因Beclin 1的表达,检测Beclin 1表达对mTOR、ULK1、LC3-B的表达以及对HCT116细胞株增殖与凋亡的影响。方法设计4条针对Beclin 1基因的干扰RNA表达载体及阴性对照载体分别瞬时转染HCT116细胞。采用RT-PCR及Western blot法筛选出1条抑制率最高的Beclin 1干扰序列后,瞬时转染HCT116细胞,检测mTOR、ULK1及LC3-B的mRNA及蛋白表达,CCK-8实验检测细胞增殖活性,流式细胞仪检测细胞凋亡情况。结果 (1)转染细胞后,BECN1-1245组mRNA及蛋白抑制率最高,达80%。(2)RT-PCR结果显示,BECN1-1245组转染细胞48 h后,与阴性对照组相比,ULK1与LC3-B mRNA表达水平降低,差异有统计学意义(P≤0.05),mTOR在两组中的表达无差异(P>0.05),Western blot法检测结果与之相同。(3)CCK-8细胞增殖实验显示,BECN1-1245组转染24、48、72 h后,细胞生存率较阴性对照组明显降低(P≤0.05)。(4)流式细胞仪检测结果显示,BECN1-1245组转染48 h后,与阴性对照组细胞相比,细胞凋亡率明显升高(P≤0.05)。结论 Beclin 1在HCT116细胞自噬中发挥重要的调控作用,抑制Beclin 1的表达,可抑制细胞的自噬活性,并能抑制细胞增殖,促进其凋亡,推测Beclin 1基因可作为结肠癌治疗的新靶点。Purpose To investigate the effects of downregulation of autophagy regulatory molecules Beclin 1 expression induced by RNA interference on the autophagy-related gene mTOR/ULK1/LC3-B and proliferation/apoptosis of HCTll6 cells. Methods HCT116 cells were transiently transfected with four specific eukaryotic expression vectors carrying shRNA targeting Beclin 1 and the negative control vectors, respectively. The most effective Beclin 1-shRNA was screened by RT-PCR and Western blot. The levels of mTOR/ULK1/LC3-B＇s mRNA and protein expression were detected by RT-PCR and Western blot. Cell proliferation and apoptosis were determined by CCK-8 detection kit and flow cytometry （FCM）. Results （ 1 ） The expression of Beclin 1 gene at mRNA and pro- tein levels was significantly lower in HCT116 cells transiently transfected with BECNI-shRNA, BECNl-1245 was the most effective, and it led to the highest inhibitory rate （80%）. （2）The mRNA and protein levels of ULK1 and LC3-B were both reduced in HCT116 cells transiently transfected with BECNl-1245 48 hours later than those in the negative control cells （P~〈0.05）, whereas no difference was found in the expression of roTOR between the two groups （P 〉0. 05）. （3） At 24, 48 and 72 hours after transfection, BECN1- 1245 group showed a lower survival rate compared with the negative control group detected by CCK-8 assay. （4） The apoptotic rate of the HCTll6 cells transfeeted with BECNl-1245 48 hours later was higher than those of the control cells evaluated by FCM （P ≤ 0. 05 ）. Conclusion Beelin 1 plays an important role in regulating autophagy in HCT116 cells, inhibition of Beclin 1 gene expression can reduce the activity of autophagy, inhibit cell proliferation and induce apoptosis. Therefore, Beelin 1 gene may be a new target of gene therapy for eolorectal cancer.

关 键 词：结肠肿瘤 自噬 RNA干扰 增殖 凋亡 BECLIN 1 HCT116 

分 类 号：R73-3[医药卫生—肿瘤] R735.35[医药卫生—临床医学]