干预HSF1影响MDR1的表达逆转结直肠癌HCT-8细胞株耐药的实验分析

Effect of HSF1 on MDR1 expression and reversal of drug resistance of colorectal cancer HCT-8 cell line

目的探讨热休克转录因子1(heat shock factor 1,HSF1)表达水平与MDR1、Cyclin D1、MMP-2、BCL-2的关系,以及对HCT-8/FU细胞株克隆和侵袭能力的影响。方法采用Western blot法检测HCT-8/FU细胞株及结肠腺癌非耐药细胞株HCT-8中HSF1及MDR1的表达。设计2条针对HSF1基因的干扰RNA表达载体及阴性对照载体,分别稳定转染HCT-8/FU细胞。采用RT-PCR及Western blot法检测各组中HSF1、MDR1的mRNA及蛋白表达量,并通过克隆实验及Transwell实验检测各组细胞生长情况和侵袭能力。通过CCK-8实验检测不同药物浓度中各组细胞的增殖活性。结果HCT-8/FU耐药细胞株中HSF1、MDR1的蛋白表达量均高于HCT-8细胞株,差异有统计学意义(P<0.01)。沉默HSF1的细胞中MDR1表达水平下降,而过表达HSF1细胞株中MDR1的表达水平增高,差异有统计学意义(P<0.01)。CCK-8细胞增殖实验结果显示沉默HSF1可提高耐药细胞对化疗药物的敏感性,其细胞增殖活性降低(P<0.05)。与空白对照组相比,沉默HSF1后Cyclin D1、MMP-2与BCL-2表达降低,可明显抑制肿瘤细胞克隆及体外侵袭能力,过表达HSF1则可增强肿瘤细胞的克隆和体外侵袭能力,差异有统计学意义(P<0.01)。结论结直肠癌中HSF1作为转录因子可促进肿瘤细胞的克隆及侵袭,同时其高表达可启动MDR1的转录与翻译,进而促进肿瘤耐药。

Purpose To investigate the effects of heat shock factor 1(HSF1)expression on the expression of MDR1,Cyclin D1,MMP-2 and BCL-2,and on the cloning and invasion ability of HCT-8/FU cell line.Methods The expression of HSF1 and MDR1 in HCT-8/FU cell line and non-drug resistant cell line HCT-8 of colon cancer was detected by Western blot.Two interfering RNA expression vectors targeting HSF1 gene and negative control vectors were designed to stably transfect HCT-8/FU cells,respectively.The mRNA and protein expression of HSF1 and MDR1 in each group was detected by RT-PCR and Western blot,and the cell growth and invasion ability of each group were detected by clone assay and Transwell assay.CCK-8 assay was used to detect the proliferation activity of each group in different drug concentrations.Results The protein expression levels of HSF1,MDR1,in colorectal cancer HCT-8/FU resistant cell lines were all higher than those in the HCT-8 cell lines,with statistical significance(P<0.01).The expression level of MDR1 was decreased in HSF1 silenced cells,while the expression level of MDR1 was increased in overexpressed HSF1 cell lines,the difference was statistically significant(P<0.01).The results of CCK-8 cell proliferation experiment showed that silencing HSF1 could improve the sensitivity of drug-resistant cells to chemotherapy drugs,and its cell proliferation activity was decreased(P<0.05).Compared with the blank control group,the expression of Cyclin D1,MMP-2 and BCL-2 decreased after HSF1 silencing,which could significantly inhibit the cloning and invasion ability of tumor cells,while the overexpression of HSF1 could enhance the cloning and invasion ability of tumor cells,with statistical significance(P<0.01).Conclusion As an transcription factor in colorectal cancer,HSF1 can promote the cloning and invasion of tumor cells,and its high expression can initiate the transcription and translation of MDR1,thus promoting drug resistance of tumor.