组蛋白H3(Ser10)磷酸化在维持肿瘤细胞恶性表型中的作用

doi:10.3969/j.issn.1004-616x.2018.02.001

癌变·畸变·突变 ›› 2018, Vol. 30 ›› Issue (2): 81-86,91.doi: 10.3969/j.issn.1004-616x.2018.02.001

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组蛋白H3(Ser10)磷酸化在维持肿瘤细胞恶性表型中的作用

黎青叶, 柳晓玲, 吴小嫩, 郭萍, 陈雯, 陈丽萍

中山大学公共卫生学院预防医学系, 广东广州 510080

收稿日期:2017-11-09 修回日期:2018-03-16 出版日期:2018-03-30 发布日期:2018-03-30
通讯作者: 陈丽萍,E-mail:chenliping_happy06@163.com E-mail:chenliping_happy06@163.com
作者简介:黎青叶,E-mail:406110671@qq.com
基金资助:
中山大学高校基本科研业务费医科青年培育项目（17ykpy13）；国家自然科学基金青年项目（31401213）

Histone H3 phosphorylation at Ser10 on maintainence of malignancy in tumor cells

LI Qingye, LIU Xiaoling, WU Xiaonen, GUO Ping, CHEN Wen, CHEN Liping

Faculty of Preventive Medicine, School of Public Health, Sun Yat-sen University, Guangzhou 510080, China

Received:2017-11-09 Revised:2018-03-16 Online:2018-03-30 Published:2018-03-30

摘要/Abstract

摘要： 目的：探讨组蛋白H3（Ser10）磷酸化在维持肿瘤细胞恶性表型中的作用。方法：采用蛋白质印迹法检测肝癌细胞株HepG2、Bel7402、SMMC-7721及人正常肝细胞L02株中H3（Ser10）的磷酸化水平，构建H3（Ser10）低磷酸化修饰突变型细胞株，通过软琼脂克隆形成试验检测下调H3（Ser10）磷酸化对肿瘤细胞恶性表型的影响。采用蛋白印迹检测改变H3（Ser10）磷酸化水平对蛋白磷酸酶2A的调控基因IGBP1（α4）表达的影响，并利用生物信息学预测IGBP1基因启动子区AP-1位点。在Bel7402和A549细胞株上构建稳定高表达AP-1和H3（Ser10）磷酸修饰位点突变型细胞株，采用双荧光素酶报告系统验证p-H3（Ser10）对α4的转录调控作用。结果：与对照组相比，肝癌细胞株中H3（Ser10）磷酸化的表达水平增加2.61倍（P < 0.05），同时发现具有癌基因功能的α4蛋白表达也上调2.0~6.3倍（P < 0.05）。H3（Ser10）位点突变的Bel7402细胞（Bel-H3S10A）中H3（Ser10）磷酸化水平明显下降，在软琼脂上形成的克隆数量减少30%。氯化镉明显诱导p-H3（Ser10）磷酸化和α4的表达（P < 0.05），且具有剂量-反应关系，提示H3（Ser10）磷酸化可能调控α4的表达。生物信息学预测发现IGBP1启动子区存在多个AP-1的结合位点。双荧光素酶报告试验结果显示，A549-AP-1-H3S10A （S10A）细胞株中H3（Ser10）磷酸化水平降低，在氯化镉处理下，α4的蛋白表达水平下降了47%（P < 0.05）。结论：组蛋白H3（Ser10）磷酸化在维持肿瘤细胞的恶性表型中起作用，可能是通过激活转录因子AP-1进而调控促癌因子α4的表达实现的。

关键词: H3(Ser10)磷酸化, α4, 肿瘤细胞, 恶性表型

Abstract: OBJECTIVE: To investigate the role and mechanism of histone H3 phosphorylation at Ser10[p-H3(Ser10)] on maintainence of malignancy in tumor cells. METHODS: Hepatic tumor cell lines including HepG2,Bel7402,and SMMC-7721 as well as human liver cell line L02 were used to detect the levels of p-H3(Ser10) based on Western blot analysis. In addition,a histone H3 mutant cell line which contained Ser10 mutated at Ala10 (Bel-H3S10A) was constructed. Soft agar assay was conducted to assess the effect of down-regulation of p-H3(Ser10) on the malignant phenotype of tumor cells. The level of histone H3 phosphorylation at Ser10 and its impact on the expression of α4 were detected by western blotting. Bioinformatics was used to identify the adaptor protein 1 (AP-1) binding site of IGBP1 promoter. The relationship between p-H3(Ser10) and the expression of α4 as well as its effect on the transcriptional level of α4 were determined by double luciferase reporter assay and western blot analysis in the constructed Bel7402 and A549 cells overexpressing AP-1and mutated histone H3. RESULTS: p-H3(Ser10) was over-expressed by 2.61 folds and α4 was increased by 2.0-6.3 folds in HCC cell lines(P < 0.05). Moreover,the level of histone H3 phosphorylation at Ser10 in Bel-H3S10A cells was decreased and formed 30% less colonies in soft agar than the control Bel-Vector cells. Cadmium chloride (CdCl₂) induced expression of p-H3(Ser10) and α4 in a dose-dependent manner(P < 0.05). However,the protein expression of α4 decreased by 47% in A549-AP-1-H3S10A cells compared to that in control cells upon CdCl₂ treatment(P < 0.05). CONCLUSION: p-H3(Ser10) played an important role in maintaining malignancy of tumor cells,perhaps through regulating the expression of α4 by activating transcription factor AP-1.

Key words: p-H3(Ser10), α4, tumor cells, malignancy

中图分类号:

R114

黎青叶, 柳晓玲, 吴小嫩, 郭萍, 陈雯, 陈丽萍. 组蛋白H3(Ser10)磷酸化在维持肿瘤细胞恶性表型中的作用[J]. 癌变·畸变·突变, 2018, 30(2): 81-86,91.

LI Qingye, LIU Xiaoling, WU Xiaonen, GUO Ping, CHEN Wen, CHEN Liping. Histone H3 phosphorylation at Ser10 on maintainence of malignancy in tumor cells[J]. Carcinogenesis, Teratogenesis & Mutagenesis, 2018, 30(2): 81-86,91.

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组蛋白H3(Ser10)磷酸化在维持肿瘤细胞恶性表型中的作用

Histone H3 phosphorylation at Ser10 on maintainence of malignancy in tumor cells

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