Msi1通过靶向调节p21对结直肠癌细胞放疗敏感性的影响  

Effects of Msi1silencing on radiosensitivity in colorectal cancer cells HCT116 by targeting p21

作　　者：高超[1] 韩春[1] 王澜[1] 刘树堂[1] 丁博月 田彦明 张翼[2] GAO Chao;HAN Chun;WANG LanLIU Shu tang;DING Bo-yue;TIAN Yan ming;ZHANG Yi(Department of Radiation Oncology,Fourth Hos pital of Hebei Medical University,Shijia zhuang 050011,P.R.China;Department of Physiology,Hebei Medical University,Shijiazhuang 050017.P.R.China)

机构地区：[1]河北医科大学第四医院放疗科,河北石家庄050011 [2]河北医科大学生理教研室,河北石家庄050017

出　　处：《中华肿瘤防治杂志》2020年第15期1209-1217,共9页Chinese Journal of Cancer Prevention and Treatment

基　　金：河北省卫计委医学科学研究重点课题计划(ZL20140107)。

摘　　要：目的放射治疗是结直肠癌综合治疗的重要组成部分,尤其是对于中晚期和术前放疗患者,目前结直肠癌对放疗敏感性较差,急需新的增敏靶点。本研究旨在探讨沉默Msi1(Musashi1)通过靶向调节p21对结直肠癌细胞放疗敏感性影响。方法蛋白质印迹法检测结直肠癌HT29、HCT116细胞及正常肠上皮细胞CCD841中Msi1蛋白表达,选择Msi1高表达的细胞系作为后续研究的靶细胞,将含有2种不同干扰序列慢病毒分别转染该细胞后,用qRT-PCR筛选出沉默效果最好的细胞株作为沉默组。实验中将结肠癌细胞分为空白组(Blank)、阴性对照组(NC)、沉默组1(Msi1-shRNA1)和沉默组2(Msi1-shRNA2)。克隆形成实验检测沉默Msi1基因对结直肠癌细胞放射敏感性的影响。MTS法检测6Gy X射线照射后结直肠癌细胞增殖情况,并计算抑制率。流式细胞术检测6Gy X射线照射后细胞凋亡情况。蛋白质印迹法检测沉默Msi1后p21蛋白表达水平,双荧光素酶实验证实Msi1与下游基因p213′-UTR相互作用。结果结直肠癌HT29、HCT116细胞中Msi1蛋白表达水平高于正常肠上皮细胞CCD841,分别为(1.000±0.181)、(1.102±0.219)和(0.106±0.051),符合正态分布,其中HCT116细胞中Msi1蛋白表达量最高(F=32.42,P=0.0006),因此选择HCT116细胞作为靶细胞进入后续实验。Msi1-shRNA1组的沉默效果优于Msi1-shRNA2组,F=68.37,P<0.001。克隆形成实验显示,经0~8Gy X射线照射后,沉默Msi1后HCT116细胞存活曲线下降,相对于空白组和阴性对照组放射增敏比SER分别为1.56和1.47,Blank组与NC组差异无统计学意义,t=2.088,P=0.1050。MTS实验结果显示,与未照射组相比,给予6Gy照射后的24、48和72h,各组细胞的增殖速度降低,并随着时间增加抑制率逐渐增加(F照射=62.16,P<0.001),Msi1-shRNA1组细胞在各时间点的抑制率均高于另外2组细胞,F时间=71.36,P<0.001。流式细胞术显示,经过6Gy照射后Msi1-shRNA1组细胞凋亡率(28.04±5.49)%高于BOBJECTIVE Radiation therapy is an important part of the comprehensive treatment of colorectal cancer,especially for patients with advanced disease and preoperative radiotherapy.Colorectal cancer is currently less senstive to radiotherapy,so new sensitization targets are urgently needed.This study aimed to explore the effects of Msi1(Musashi1)silencing on radiosensitivity in colorectal cancer cells by targeting p21.METHODS The protein expression of Msi1 in normal colon epithelial cell line CCD841 and colorectal cancer cells HT29 and HCT116 were determined by Western blot.The cell line with high expression of Msi1 was selected as the target cell for subsequent research,the lentivirus containing two different interference sequences were transfected into the cells,and the cell line with the best silencing effect was screened by qRT-PCR.In the experiment,colon cancer cells were divided into blank group,negative control(NC)group,silent group1(Msi1-shRNA1)and silent group2(Msi1-shRNA2).Clone formation assay was performed to examine the effect of silencing Msi1 gene on radiosensitivity of colorectal cancer cells.MTS assay was used to detect the proliferation of colorectal cancer cells after 6 Gy X-ray irradiation,and the inhibition rate was calculated.Flow cytometry analysis was used to detect apoptosis after 6 Gy X-ray irradiation,Western blot was used to detect the expression of p21 protein after silencing Msi1,and the binding site of Msi1 to p213′-UTR was examined by Luciferase reporter assay.RESULTS(1)The expression of Msi1 protein in colorectal cancer HT29(1.000±0.181)and HCT116(1.102±0.219)cells was significantly higher than that in normal intestinal epithelial cells CCD841(0.106±0.051)(F=32.42,P=0.0006),data accorded with normal distribution.Due to the highest expression of Msi1 protein,HCT116 cells were selected as targets cell line.The silencing effect of the Msi1-shRNA1 group was better than that of the Msi1-shRNA2 group(F=68.37,P<0.0001).(2)Clone formation assay showed that the survival curve after irradi

关 键 词：结直肠癌 Msi1 放疗敏感性 P21 凋亡 

分 类 号：R735.35[医药卫生—肿瘤]