缺氧微环境SIRT1亚细胞定位对结直肠癌细胞凋亡的影响及其机制研究

Effect and Its Mechanism of SIRT1 Subcellular Location on Apoptosis of Colorectal Carcinoma in Hypoxic Microenvironment

目的:探究缺氧微环境SIRT1亚细胞定位对结直肠癌细胞凋亡的影响及其分子机制。方法:将编码过表达野生型SIRT1以及核定位序列(nuclear localization sequence,NLS)突变型SIRT1(SIRT1NLSmt)的慢病毒载体转染人类结肠癌HCT116细胞株,经嘌呤霉素筛选获得稳定过表达野生型SIRT1细胞株(LV-SIRT1细胞)和细胞质定位的NLS突变型SIRT1细胞株(LV-SIRT1NLSmt细胞),通过观察慢病毒载体编码的SIRT1-GFP融合蛋白的荧光定位,明确稳定转染细胞中外源性SIRT1的亚细胞定位。利用real-time PCR、Western blot法对分离提取的核-质蛋白进行检测,证实外源性SIRT1的表达和亚细胞定位情况。利用CCK-8细胞毒性实验、流式细胞术检测和TUNEL染色比较缺氧(1%O2)处理前后LV-SIRT1和LV-SIRT1NLSmt细胞存活或凋亡情况,Western blot法检测凋亡相关蛋白p53、ac-p53(K382)、Bcl-2、Bax、caspase-3和cleaved caspase-3表达水平。结果:Western blot、real-time PCR和免疫荧光染色结果显示稳定转染细胞均存在外源性SIRT1的过表达,NLS突变可导致SIRT1NLSmt富集于细胞质中;与亲本细胞HCT116和LV-SIRT1NLSmt细胞相比,LV-SIRT1细胞对缺氧的耐受能力最差、细胞凋亡水平最高,凋亡相关蛋白p53、Bax、caspase-3、cleaved caspase-3表达水平显著升高,ac-p53(K382)和Bcl-2表达水平显著下降,且LV-SIRT1细胞的胞核ac-p53下降最为显著。结论:在缺氧微环境中,细胞核定位的SIRT1通过影响p53的去乙酰化水平促进结直肠癌细胞凋亡。

Objective:To explore the effect of subcellular localization of SIRT1 on apoptosis of colorectal carcinoma cells under hypoxic microenvironment and its molecular mechanism.Methods:Human colon carcinoma HCT116 cells overexpressing wild-type SIRT1(LV-SIRT1 cells)and SIRT1 with nuclear localization sequences/NLSs(LV-SIRT1NLSmt cells)were obtained via transfection with lentiviral vectors carrying the corresponding coding sequences and screening with puromycin,respectively.The subcellular localizations of exogenous SIRT1 were observed by the fluorescence signals of SIRT1-GFP fusion proteins encoded by lentiviral vectors.The mRNA and protein expression levels of SIRT1 were analyzed by real-time PCR and Western blot assay.Both cytoplasmic and nuclear extractions were used to analyze the subcellular localization of exogenous SIRT1 by Western blot assay.CCK-8 cytotoxic assay,flow cytometry analysis and TUNEL staining were performed to compare the rates of cell survival or apoptosis among different cell groups after hypoxia(1%O2)treatment.The expression levels of apoptosis-related proteins,p53,ac-p53(K382),Bcl-2,Bax,caspase-3 and cleaved caspase-3,were analyzed by Western blot assay.Results:Both LV-SIRT1 and LV-SIRT1NLSmt cells had stable overexpression of exogenous SIRT1,while the mutation of NLSs led to the enrichment of SIRT1NLSmt in the cytoplasm.Compared with parental cells,HCT116,and LV-SIRT1NLSmt cells,LV-SIRT1 cells exhibited the worst tolerance to hypoxic treatment,the highest level of apoptosis,the most significantly increased protein expression levels of p53,Bax,caspase-3 and cleaved caspase-3,and the most significantly decreased protein expression levels of Bcl-2 and ac-p53(K382).And there was the most significantly decreased ac-p53 protein expression level in the nucleus of LV-SIRT1 cells.Conclusion:Nuclear localization of SIRT1 promotes apoptosis in colorectal carcinoma cells by affecting the deacetylation level of p53 under hypoxic microenvironment.