乏氧对人口腔鳞状细胞癌细胞中叉头蛋白P3表达的影响及其机制

doi:10.13481/j.1671-587x.20170306

吉林大学学报(医学版) ›› 2017, Vol. 43 ›› Issue (03): 491-495.doi: 10.13481/j.1671-587x.20170306

乏氧对人口腔鳞状细胞癌细胞中叉头蛋白P3表达的影响及其机制

闫风琴¹, 范如意², 王方正¹, 王磊¹, 叶智敏¹, 傅真富¹, 王建秋²

1. 浙江省肿瘤医院放疗科十九病区, 浙江杭州 310022;
2. 杭州师范大学医学院生物化学与分子生物学教研室, 浙江杭州 310036

收稿日期:2016-12-09 出版日期:2017-05-28 发布日期:2017-06-01
通讯作者: 王建秋,副教授,硕士研究生导师(Tel:0571-28865671,E-mail:jianqiuw@hznu.edu.cn) E-mail:jianqiuw@hznu.edu.cn
作者简介:闫风琴(1978-),女,河南省卫辉市人,主治医师,医学博士,主要从事头颈癌放射治疗方面的研究。
基金资助:
国家自然科学基金资助课题（81301850）；浙江省教育厅科研项目资助课题（Y201328812）

Effect of hypoxia on forkhead box P3 expression in human oral squamous cell carcinoma cells and its mechanism

YAN Fengqin¹, FAN Ruyi², WANG Fangzheng¹, WANG Lei¹, YE Zhimin¹, FU Zhenfu¹, WANG Jianqiu²

1. Department of Radiation Oncology, Zhejiang Provincial Cancer Hospital, Hangzhou 310022, China;
2. Department of Biochemistry and Molecular Biology, School of Medical Sciences, Hangzhou Normal University, Hangzhou 310036, China

Received:2016-12-09 Online:2017-05-28 Published:2017-06-01

摘要/Abstract

摘要： 目的：观察乏氧对人口腔鳞状细胞癌（OSCC）细胞中叉头蛋白P3（FOXP3）表达的影响，阐明乏氧调控FOXP3表达的表观遗传学机制。方法：选取人OSCC细胞株FaDu和OECM-1细胞，常氧和乏氧条件下培养18h。采用实时定量PCR方法检测细胞中FOXP3 mRNA的相对表达水平，Western blotting 法检测FaDu和OECM-1细胞中FoxP3蛋白表达水平，染色质免疫沉淀-定量PCR（ChIP-qPCR）法检测FOXP3基因启动子上组蛋白修饰H3K4乙酰化（H3K4ac）、H3K4三甲基化（H3K4me3）和H3K27三甲基化（H3K27me3）水平。采用组蛋白去乙酰化酶3（HDAC3）基因沉默的FaDu细胞，以实时定量PCR和ChIP-qPCR法检测FaDu细胞中HDA3 mRNA相对表达水平和FOXP3mTNA表达抑制率。结果：与常氧条件比较，乏氧条件下FaDu和OECM-1细胞中FOXP3 mRNA相对表达水平均明显降低，分别下降65.6%（P<0.01）和75.7%（P<0.01）；Western blotting检测，乏氧条件下FaDu和OECM-1细胞中FOXP3蛋白表达水平明显低于常氧条件下；染色质免疫沉淀实验，与常氧条件比较，乏氧条件下FaDu细胞中FOXP3基因启动子上的H3K4ac和H3K4me3水平明显降低（P<0.01），而H3K27me3水平无明显差异。与无HDAC3基因沉默的FaDu细胞比较，HDAC3基因沉默的FaDu细胞中缺氧诱导的FOXP3启动子上H3K4ac和H3K4me3表达抑制率明显降低（P<0.05），缺氧诱导的FOXP3mRNA相对表达抑制率降低（P<0.05）。结论：乏氧可通过HDAC3介导的FOXP3基因启动子上H3K4ac水平下调而抑制OSCC细胞中FOXP3的表达。

关键词: 乏氧, 口腔肿瘤, 叉头蛋白P3, 鳞状细胞, 组蛋白修饰, 癌

Abstract: Objective: To investigate the effect of hypoxia on the expression of forkhead box P3 (FOXP3) in human oral squamous cell carcinoma (OSCC) cells, and to clarify its possible epigenetic mechanism. Methods: Two kinds of OSCC cell lines, FaDu and OECM-1, were cultured under normoxic or hypoxic conditions for 18 h. The relative expression levels of FOXP3 mRNA and protein in the cells were detected by Real-time RT-PCR and Western blotting method. The histone modification levels on the FOXP3 gene promoter, including acetylation of histone 3 lysine 4 (H3K4ac), trimethylation of histone 3 lysine 4 (H3K4me3) and lysine 27 (H3K27me3), were analyzed by Chromatin Immunocipitation (ChIP) and quantitative PCR (ChIP-qPCR). The relative expression levels of histone deacetylase 3 (HDAC3) mRNA and inhibitory rates of FOXP3 mRNA expression in the HDAC3-knockdown FaDu cells were investigated by Real-time qPCR and ChIP-qPCR. Results: Compared with normoxic condition, the relative expression levels of FOXP3 mRNA in FaDu and OECM-1 cells under hypoxic condition were decreased by 65.6% and 75.7% (P<0.01). The Western blotting results indicated that compared with normoxic condition, the expression levels of FOXP3 protein in FaDu and OECM-1 cells under hypoxic condition were decreased. The ChIP experiment results showed that compared with normoxic condition, the levels of H3K4ac and H3K4me3 on FOXP3 gene promoter in FaDu cells were decreased under hypoxic condition (P<0.01), while the H3K27me3 level was not changed.In HDAC3-knockdown FaDu cells, compared with control cells, the inhibitory rates of the expressions of H3K4ac and H3K4me3 on FOXP3 gene promoter under hypoxia condition were decreased (P<0.05), so did expressions the FOXP3 mRNA expression (P<0.05). Conclusion: Hypoxia could suppress the expression of FOXP3 by HDAC3-mediated down-regulation of H3K4ac on FOXP3 gene promoter in the human OSCC cells.

Key words: hypoxia, oral neoplasms, forkhead box P3, histone modification, carcinoma, squamous cells

中图分类号:

R739.8

闫风琴, 范如意, 王方正, 王磊, 叶智敏, 傅真富, 王建秋. 乏氧对人口腔鳞状细胞癌细胞中叉头蛋白P3表达的影响及其机制[J]. 吉林大学学报(医学版), 2017, 43(03): 491-495.

YAN Fengqin, FAN Ruyi, WANG Fangzheng, WANG Lei, YE Zhimin, FU Zhenfu, WANG Jianqiu. Effect of hypoxia on forkhead box P3 expression in human oral squamous cell carcinoma cells and its mechanism[J]. Journal of Jilin University Medicine Edition, 2017, 43(03): 491-495.

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乏氧对人口腔鳞状细胞癌细胞中叉头蛋白P3表达的影响及其机制

Effect of hypoxia on forkhead box P3 expression in human oral squamous cell carcinoma cells and its mechanism

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