miR-133b调控GSTP1表达降低结肠肿瘤细胞在低营养环境下的生存能力

Viability of Colon Tumor Cells in Insufficient-nutritional Condition is Reduced by MiR-133b Through Regulating

中文关键词:  GSTP1 miR-133b 结直肠癌 低营养环境

英文关键词:GSTP1 MiR-133b Colon cancer Insufficient-nutritional condition

中文摘要:

      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pcDNA3.1GSTP1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pGL3GSTP13′UTR，共转染该miR与pGL3GSTP13′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pcDNA3.1GSTP1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miR133b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miR133b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miR133b无显著影响。此外，在低营养环境下，过表达miR133b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miR133b能够通过对GSTP1的抑制作用削弱结肠肿瘤细胞的生存能力。

英文摘要:

      【Abstract】 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 Realtime PCR and western blot were used to detect the expression level of GSTP1 in colon tumor tissues. pcDNA3.1GSTP1 was constructed and transfected into human colon tumor cell line HCT116. The apoptosis rate and viability of HCT116 were measured. TargetScan and MicroCosm Targets programs were used to measure GSTP1 3′UTR and identify GSTP1targeting miR. pGL3GSTP13′UTR was constructed and transfected with the selected miR into human kidney epithelial cell line HEK293. Dualluciferase 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 pcDNA3.1GSTP1 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. Overexpression of GSTP1 decreased apoptosis and increased viability of HCT116 in insufficientnutritional condition. miR133b could target GSTP1 3′UTR and repressively regulate GSTP1 expressions in HCT116. The overexpression of miR133b rapidly decreased viability and increased ROS level in HCT116 in insufficientnutritional condition. But such effects were absent in normal nutritional condition. In addition, in insufficient nutrition condition, when the HCT116 overexpress miR133b and GSTP1 at the same time, the extent of viability decrease and ROS level increase are shortened. Conclusion In insufficientnutritional condition, miR133b decreases viability of colon tumor cells by repressively regulating GSTP1 expressions.