RNA干扰抑制γ突触核蛋白的表达对人结肠癌细胞株增殖和侵袭能力的影响  

Construction of lentiviral vector of siRNA specific for γ-synuclein and its inhibition effect on colorectal carcinoma cell line SWl116

作　　者：黄峰[1] 许少华[1] 叶青[1] 郑秋红[1] 许扬梅[1] 刘沁颖[1] 

机构地区：[1]福建省肿瘤医院胃肠肿瘤外科,福州350014

出　　处：《中华胃肠外科杂志》2016年第4期446-452,共7页Chinese Journal of Gastrointestinal Surgery

摘　　要：目的探讨抑制γ突触核蛋白（SNCG）基因表达对结肠癌细胞株生长及侵袭能力的影响。方法针对SNCG基因的mRNA序列（GENBANK：No．NM003087．2），设计合成干扰SNCG基因表达的RNA（siRNA）有效靶序列及其DNA，与慢病毒骨架质粒GV115（hU6-MCS-CMV—EGFP）连接并包装．获得实验组慢病毒LV-SNCG-RNAi-EGFP，并用相同步骤构建阴性对照组慢病毒LV-SNCG-NCIEGFP。用两种重组慢病毒分别感染人结肠癌SW1116细胞（实验组：RNAi组，阴性对照组：NC组），荧光显微镜下观察感染后shRNA荧光标签（EGFP）的表达情况；利用实时定量聚合酶链反应（Real．timePCR）检测RNAi组与NC组细胞中SNCG基因表达mRNA的干扰效果；以野生型SW1116细胞为空白对照（野生型组），用Westernblot方法检测RNAi组、NC组和野生型组细胞中SNCG基因表达蛋白的干扰效果；CCK．8细胞增殖实验、软琼脂克隆形成实验和Transwell侵袭实验．用于评价RNA干扰SNCG基因表达对RNAi组与NC组细胞的体外生长及侵袭能力的抑制作用。结果成功构建并获得了稳定表达重组慢病毒的SW1116细胞株，表达慢病毒LV-SNCG-RNAi-EGFP及LV-SNCG-NC-EGFP滴度均为8×10^8Tu／ml。RNAi组SNCG基因相对表达水平（2-△△△）为0．114±0．030，低于于对照NC组的SNCG基因相对表达水平（2-△△Q：1．009±0．161），差异有统计学意义（P=0．009）；RNAi组SNCG基因干扰效率达到76．8％。RNAi组的SNCG蛋白相对表达量为12．001±2．884，低于NC组（蛋白相对表达量：32．445±4，731）和野生型组（蛋白相对表达量：34．308±6．920），差异有统计学意义（P=0．018，P=0．020）。CCK-8细胞增殖实验显示，干扰SNCG基因表达后，RNAi组细胞的体外增殖能力从48h开始受到明显抑制，并持续到120h，与NC组及野生型组差异具有统计学意义（P=0．036）。RNAi组克隆明显偏小，RNAi组平均直径（0．582±0．103）mm，低�Objective To construct a lentiviral vector carrying the γ-synuclein （SNCG） gene and establish a human colorectal carcinoma cell line SW1116 stably expressing this gene, and theninvestigate the inhibition of the growth and invasion capacity of SWIll6 cells. Methods RNA interference fragment was designed according to the SNCG sequence （C, enBank： No.NM003087.2）, and then SNCG RNAi effective target genes were screened. After the Oligo DNA of target sequences was synthesized, the lentiviral vectors carrying LV-SNCG-RNAi-EGFP （RNAi group） and LV-SNCG-NC- EGFP （NC group） were constructed and packaged to produce lentivirus venom. The supematants of different virus-producing cells were used to transfect SWII16 cells respectively. Wild SWII16 cells were used as blank control （CON group） EGFP fluorescence was detected by fluorescent microscopy and the differential expression of SNCG mRNA and protein was detected by real-time PCR and Western blot. CCK-8, soft agar assay and Transwell chamber were employed to estimate the inhibiting effect on growth and invasion of SW1116 respectively. Results Recombinant lentiviral vectors respectively carrying the SNCG-RNAi-EGFP and SNCG-NC-EGFP were successfully constructed and the supernatants of lentivirns could effectively infect SWlll6 cells. The titer of the virus carrying LV-SNCG-RNAi-EGFP or LV- SNCG-NC-EGFP was 8x10s TU/ml. Real-time PCR and Western blot confirmed that compared with the NC group, SNCG-RNAi group had lower SNCG expression （1.009 ± 0.161 vs. 0.114 ± 0.030, P = 0.009）, and showed tremendous silencing effect as 76.8%（P 〈 0.05）. SNCG protein expression was also significantly reduced （RNAi： 12.001 ± 2.884, NC：32.443 ~： 4.731, CON：34.308 ± 6.920, P 〈 0.05）. After SNCG knockdown, the number of proliferation cells was obviously reduced at 48, 72, 96 and 120 hours respectively （P=0.036）. In soft agar assay, clones in RNAi group were smaller[RNAi： （0.582 ± 0.103） mm, NC：（1.863 ＋ 0.316） mm, CON：（1.749 f

关 键 词：结肠肿瘤 SNCG基因 慢病毒载体 RNA干扰 基因转染 

分 类 号：R735.35[医药卫生—肿瘤]