作　　者： (李昂）; (杨川）; (胡敏）;

机构地区： 四川大学生命科学学院,成都610036

出　　处： 《四川大学学报（医学版）》 2017年第5期699-704,共6页

摘　　要： 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 determine the expression level of GSTP1 in colon tumor tissues at different stages and the effects of GSTP1 expression on colon tumor cells, and to identify miR that regulate the expression of GSTP1. Methods Real-time PCR and western blot were used to detect the expression level of GSTP1 in colon tumor tissues, pcDNA3.1-GSTP1 was constructed and transfected into human colon tumor cell line HCTll6. The apoptosis rate and viability of HCTll6 were measured. TargetScan and MicroCosm Targets programs were used to measure GSTP1 a＇UTR and identify GSTPl-targeting miR. pGL3-GSTP1-3＇UTR was constructed and transfected with the selected miR into human kidney epithelial cell line HEK293. Dual-luciferase reporter assay were performed to detect the regulation effect of the selected miR on GSTP1 expressions. The selected miR was transfected into HCT116, and their levels of GSTP1 expression and reactive oxygen species （ROS） were measured. The selected miR and pcDNA3. 1-GSTP1 were then transfected together into HCT116. The viability and ROS level of the transfected cells were measured. Results The expression levels of GSTP1 increased in colon tumor tissues at all stages. Over-expression of GSTP1 decreased apoptosis and increased viability of HCT116 in insufficient-nutritional condition, miR-133b could target GSTP1 a＇UTR and repressively regulate GSTP1 expressions in HCTll6. The over-expression of miR-133b rapidly decreased viability and increased ROS level in HCT116 in insufficient- nutritional condition. But such effects were absent in normal nutritional condition. In addition, in insufficient- nutrition condition, when the HCTll6 over-express miR-133b and GSTP1 at the same time, the extent of viability decrease and ROS level increase are shortened. Conclusion In insufficient-nutritional condition, miR-133b decreases viability of colon tumor cells by repressively regulating GSTP1 expressions.

关 键 词： 结直肠癌 低营养环境