p65基因序列shRNA慢病毒载体构建及其作用功能的初步研究  

Research of the p65 gene function in the prostate cancer cell by the obtaining of shRNA sequences blocking the expression of nuclear factor kappa- B （p65） stably and construction of lentivirus vector

作　　者：黄海[1] 杜涛[1] 黄健[1] 林天歆[1] 张彩霞[1] 董文[1] 尹心宝[1] 郭正辉[1] 许可慰[1] 江春[1] 韩金利[1] 

机构地区：[1]中山大学附属第二医院泌尿外科,广州510120

出　　处：《中华泌尿外科杂志》2010年第6期386-390,共5页Chinese Journal of Urology

基　　金：广东省自然科学基金资助项目（001356,05100980,05300720）;广东省医学科学基金资助项目（A2008178）

摘　　要：目的 建立稳定阻断前列腺癌细胞株LNCaP中核因子NF-κB（p65）表达的shRNA序列,构建慢病毒载体,检测p65在前列腺癌细胞中的作用功能.方法 根据p65基因信息,设计siRNA1、siRNA2、siRNA3针对p65基因cds区的siRNA序列,组建shRNA对应的4对互补单链DNA将合成的序列插入空载体pSIH1-H1-copGFP shRNA Vector中转染前列腺癌细胞,采用RTPCR方法检测不同序列片断对p65 mRNA的抑制效率,免疫印迹方法检测其对p65蛋白表达的抑制效率.将获得的慢病毒载体感染LNCaP细胞,设对照组、空转组.采用MTT、流式细胞术、Transwell等方法检测p65的生物学作用.结果 设计的3条针对p65序列中,第3条序列对前列腺癌细胞株中p65 mRNA的干扰效率为59%,对蛋白表达的抑制率达81%.目的序列位于p65（NM_021975）的1096～1113,茎环序列为5＇-GATCC GCCCTATCCCTTTACGTCATTCAAGAGAT-GACGTAAAGGGATAGGGCTTTTTG-3＇.转染细胞后,细胞可以稳定低表达p65.MTT检测实验组细胞96 h内未出现对数增殖,生长能力低于对照组和空转组.流式细胞检测实验组G0～G1期细胞百分率为61.49%,高于对照组的44.89%和空转组的41.52%;而S期细胞百分率为28.58%,低于对照组的47.36%和空转组的46.10%,差异有统计学意义（P＜0.05）.Transwell检测实验组透过细胞数为（16.5000±6.62076）个,低于对照组和空转组[（45.6333±13.54159）个,（36.8333±5.68412）个],差异有统计学意义（P＜0.05）.结论 成功获得稳定阻断前列腺癌细胞株LNCaP中p65表达的shRNA序列,并构建慢病毒载体;初步验证了p65在前列腺癌细胞增殖、侵袭和转移中的促进作用.Objective To obtain shRNA sequences that can stably block the expression of Nuclear Factor kappa- B （p65） in the prostate cancer cell line LNCaP and construct the lentivirus vector.And validate the gene function of p65 in the cell line. Methods According to p65 genetic information, we design siRNA1, siRNA2, siRNA3 those three siRNA sequences targeting the ods area of p65 gene and then form the corresponding four pairs of complementary single strand DNA of shRNA, including the sense strand and the antisense strand. The synthetic shRNA sequence was inserted into the empty pSIH1-H1-copGFP shRNA Vector, and after transfecting the prostate cancer cells , the inhibitory effect of p65 mRNA by different sequences was detected through real-time PCR, and the inhibitory effect of p65 protein expression was detected by Western-blotting. Thus we can obtain highly effective shRNA sequences in the inhibition of p65 in prostate cancer cells. MTT, flow cytometry, transwell were chosen to test the cell growth, migration and invasive power in vitro to compare the difference of the experimental group, control group and negative group. Results The third shRNA sequence had the best inhibitory effect and the inhibitory effect of p65 mRNA in prostate cancer cell line was 59 % and the protein was 81%. It＇s position locates in p65 （NM_021975 ） 1096-1113 and it＇s stemloop sequence is 5＇-GATCCGCCCTATCCCTTTACGTCATTCAAGAGATGACGTAAAGGGATAGGGCTTTTTG-3＇. After transfecting, the prostate cancer cell line had the low expression of p65 stably. Through MTT, we got the growth curve, which showed that the growth ability of experimental group was significantly decreased compared with the control group and the Logarithmic growth didn＇t appear in the first 96 hours. Flow cytometry test displayed that the percentage of G0-G1-phase cells in experimental group was 61.49%, and the control group was 44.89%, idle group was 41.52%, which was increasing oberviously. The S-phase cells in the experimental group was 28.58%, compared with the 47.36

关 键 词：前列腺肿瘤 癌 核因子ΚB 

分 类 号：R737[医药卫生—肿瘤]