| 崔竹青,余沈桐,杨 桐,周 汝,张 静.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[J].,2020,(13):2411-2417 |
| 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 |
| 投稿时间：2019-12-23 修订日期：2020-01-18 |
| DOI：10.13241/j.cnki.pmb.2020.13.003 |
| 中文关键词: 结肠癌 缺氧 SIRT1 亚细胞定位 细胞凋亡 |
| 英文关键词: Colorectal carcinoma Hypoxia SIRT1 Subcellular location Apoptosis |
| 基金项目:肿瘤生物学国家重点实验室自主研究课题（CBSKL2017Z21）；国家自然科学基金项目（ 81572545） |
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| 中文摘要: |
| 摘要 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。 |
| 英文摘要: |
| ABSTRACT 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. |
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