在结肠癌细胞中P53突变调控KLF5功能及表达变化的实验研究  

P53 mutation regulate the change of function and expression of KLF5 in the colon cancer cell

作　　者：曹杰智 赖东明[2] 伍衡[2] 褚忠华[2] 曾育杰[2] 

机构地区：[1]广州白云区中医医院外二科,510470 [2]广州中山大学孙逸仙纪念医院胃肠外科,510120

出　　处：《岭南现代临床外科》2017年第4期410-414,共5页Lingnan Modern Clinics in Surgery

基　　金：广东省医学科研基金(A2015554)

摘　　要：目的探讨在结肠癌细胞株中P53基因突变对KLF5基因功能及表达变化的影响。方法通过对常见结肠癌细胞株的筛选,选取P53野生型及KLF5基因蛋白表达相对较低的细胞株作为研究对象,应用质粒构建过表达载体转染目的细胞,运用PCR及Western blot技术进行验证,再用细胞迁移、侵袭、增殖实验观察结肠癌细胞株中P53突变与否对KLF5基因功能及表达的影响。结果通过应用Western blot技术对常见的7株结肠癌细胞株中KLF5基因蛋白表达以及应用Sanger测序对细胞株中P53突变情况的筛选,选取结肠癌细胞株RKO作为研究对象,在成功构建P53^(R175H)过表达载体及KLF5干扰和过表达载体,并转染到RKO细胞株中,并行Western-blot验证后,运用细胞迁移、侵袭及增殖实验,发现在没有P53^(R175H)突变的RKO结肠癌细胞株中,KLF5的过表达能抑制细胞的迁移、侵袭及增殖的能力,而敲除KLF5的表达后,可以发现细胞的迁移、侵袭及增殖能力明显增加;而在P53^(R175H)突变后的结肠癌细胞株RKO中,KLF5的过表达则会导致RKO的迁移、侵袭及增殖能力明显增强,而敲除KLF5的表达后迁移、侵袭及增殖能力下降。同时也发现RKO细胞中P53的突变会导致KLF5的表达水平的下降。结论在高表达P53突变的结肠癌细胞株RKO中,KLF5的蛋白表达水平下降,而且其基因功能则从抑癌向促癌方向转换。Objective To investigate P53 mutation impact on the function and expression of KLF5 in colon cancer cell. Methods Take the colon cancer cell lines RKO as the research object,which was both lower KLF5 expression and P53 wild type,chosen from the common colon cancer cells by Western blot and Sanger sequencing. Construct the over-expression/knock-down cell RKO-PCDNAKLF5 and RKO-PCDNA-sh KLF5,RKO-P53^（R175H）-KLF5 and RKO-P53^（R175H）-sh KLF5 by the plasmid and verified by Western blot. Transwell assay was used to examine the invasion and metastasis ability. MTS assay was used to examine the proliferation ability. Results In the colon cancer cell lines RKO which was P53 wild type,Transwell assays revealed that the migratory and invasive capacities were inhibited by over-expression of KLF5,as well proliferation ability. And when the KLF5 was silent,the capacities of migratory and invasive,as well as proliferation ability were enhanced obviously. On the contrast with this result,the capacities of migratory,invasive and proliferation in the RKO-P53^（R175H）cell with over-expression KLF5 or silent KLF5 showed the opposite conclusion. At the same time,the protein level expression of KLF5 in the RKO-P53^（R175H）cell was declined,contrast to the RKO cell with P53 wild type. Conclu-sion In colon cancer cell lines RKO with high expression of P53 mutation,the protein expression of KLF5 levels dropped,and the gene function transformed from a tumor suppressor to the tumor promoter.

关 键 词：结肠癌 P53突变 KLF5 

分 类 号：R817[医药卫生—影像医学与核医学]