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目的探究GSTP1在不同阶段结肠癌组织中的表达水平,并查找可能对GSTP1表达产生调控作用的微小分子RNA(miR)及其对结肠肿瘤细胞的影响。方法利用实时定量PCR和蛋白印迹分析检测GSTP1在结肠癌组织中的表达水平;构建载体pcDNA3.1-GSTP1并转染到培养的人类结肠癌细胞系HCT116中,检测细胞的凋亡率与活性水平;利用TargetScan和MicroCosm Targets程序分析GSTP1的3′UTR序列并查找能够结合GSTP1的miR,构建载体pGL3-GSTP1-3′UTR,共转染该miR与pGL3-GSTP1-3′UTR到人类肾脏上皮细胞系HEK293中,荧光素酶报告分析验证该miR对GSTP1表达的靶向调控作用;转染该miR到HCT116中,检测GSTP1表达水平与细胞的活性氧(ROS)水平;共转染该miR与pcDNA3.1-GSTP1到HCT116中,检测细胞的ROS水平与活性水平。结果GSTP1在不同阶段结肠癌样本中的表达水平均上升,在低营养环境下,高表达GSTP1的HCT116细胞凋亡率下降,细胞活性上升。而miR-133b在GSTP1的3′UTR序列区域有特异性结合位点,在转录后水平下调GSTP1的表达。在低营养环境下,过表达miR-133b的HCT116细胞活性水平显著下降,ROS水平显著上升;在正常营养环境下,高表达miR-133b无显著影响。此外,在低营养环境下,过表达miR-133b的HCT116细胞同时过表达GSTP1,则细胞活性水平下降程度和ROS水平上升程度均减小。结论低营养环境下miR-133b能够通过对GSTP1的抑制作用削弱结肠肿瘤细胞的生存能力。
Objective To investigate the expression of GSTP1 in colon cancer tissues at different stages and to find out the possible effect of microRNA (miR) on the expression of GSTP1 and its effect on colon tumor cells. Methods The expression of GSTP1 in colon cancer tissues was detected by real-time quantitative PCR and Western blotting. The vector pcDNA3.1-GSTP1 was constructed and transfected into human colon cancer cell line HCT116 to detect the apoptosis rate and the activity level ; Using the TargetScan and MicroCosm Targets program to analyze the 3’UTR sequence of GSTP1 and find the miRs capable of binding to GSTP1, the vector pGL3-GSTP1-3’UTR was constructed and the miR and pGL3-GSTP1-3’UTR were co-transfected into human renal epithelial cells Department of HEK293, luciferase reporter assay to verify the targeted regulation of GSTP1 expression by this miR; transfected this miR into HCT116 to detect GSTP1 expression level and reactive oxygen species (ROS) levels; cotransfection of this miR and pcDNA3 .1-GSTP1 to HCT116, the ROS level and the activity level of the cells were detected. Results The expression level of GSTP1 in colon cancer samples at different stages increased. Under low nutritional conditions, the apoptosis rate of HCT116 cells highly expressing GSTP1 decreased and the cell activity increased. However, miR-133b has a specific binding site in the 3’UTR region of GSTP1 and down-regulates the expression of GSTP1 at the post-transcriptional level. Under low nutrient conditions, the activity of HCT116 cells overexpressing miR-133b significantly decreased and the level of ROS significantly increased. In normal nutrition, miR-133b overexpression did not significantly affect cell viability. In addition, under low-nutrition conditions, HCT116 cells overexpressing miR-133b overexpress GSTP1 at the same time, the decrease of cell viability and ROS level decrease. Conclusion miR-133b can weaken the viability of colon tumor cells through the inhibition of GSTP1 in low nutrient environment.