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MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制

吴婷^*, 黄小花

厦门大学医学院基础医学部，厦门 361005

摘要

多发性内分泌肿瘤1型(multiple endocrine neoplasia type 1, MEN1)是一种常染色体显性遗传的肿瘤综合征，患者常表现出多发性的内分泌器官肿瘤，包括垂体瘤、甲状旁腺瘤和胰岛瘤。抑癌基因Men1的突变导致MEN1的发生，其编码的蛋白为核蛋白menin。Menin可以抑制包括胰岛β细胞在内的内分泌细胞增殖。本文探讨menin通过抑制细胞增殖来抑制MEN1胰岛瘤的可能机制。在基因芯片的分析中，喂养它莫西酚(tamoxifen)14 d后诱导条件敲除Men1的小鼠胰岛中cyclin B2表达上升。免疫荧光的实验显示在小鼠MEN1胰岛瘤的组织切片中，cyclin B2表达量显著增加。细胞水平的研究显示，在Men1敲除的细胞中，cyclin B2表达量上升。以第10位丝氨酸磷酸化的组蛋白H3 (phospho-H3S10)抗体对细胞进行免疫荧光实验，显示Men1敲除的细胞中处于有丝分裂的细胞数增加。以5 × 104的密度接种细胞，在第2天、第4天和第6天的时候分别对细胞计数，显示Men1敲除的细胞增殖加快。相反地，用shRNA敲低细胞中的cyclin B2，则细胞中处于有丝分裂的细胞数减少，细胞增长减慢。染色质免疫沉淀(chromatin immunoprecipitation, ChIP)分析显示，menin影响了cyclin B2启动子区组蛋白H3第4位赖氨酸的三甲基化水平及组蛋白H3的乙酰化水平，但不影响组蛋白H3第9位赖氨酸和第27位赖氨酸的三甲基化水平。上述结果提示menin可能通过组蛋白修饰而抑制cyclin B2的表达从而抑制了MEN1胰岛瘤的生长。

关键词： 胰岛瘤; cyclin B2; 有丝分裂; 细胞增殖; menin

分类号：R335+.6

[The role and mechanism of high expression of cyclin B2 in MEN1 insulinoma.] [Article in Chinese]

WU Ting^*, HUANG Xiao-Hua

Department of Basic Medical Sciences, Medical College, Xiamen University, Xiamen 361005, China

Abstract

Multiple endocrine neoplasia type 1 (MEN1) is a dominantly inherited tumor syndrome characterized by development of various combinations of tumors in multiple endocrine glands, including the pituitary, parathyroid or pancreas. MEN1 results from mutations in tumor suppressor gene Men1, which encodes nuclear protein menin. Menin has been shown to preferentially repress cell proliferation in endocrine tissues including pancreatic beta cells. Herein, the present study was to explore the potential mechanisms underlying menin in repressing cell proliferation in mice MEN1 insulinoma. In the Gene Set Enrichment Analysis (GSEA), Ccnb2 (encoding cyclin B2) was up-regulated in pancreatic islets of Men1-excised mice after 14-day tamoxifen-feeding. Immunofluorescence with antibody against cyclin B2 revealed that the expression of cyclin B2 was greatly increased in MEN1 insulinoma. In Men1−/− cells, Men1 ablation leaded to an increase in cyclin B2 expression. Immunofluorescent staining by phospho-H3S10 antibody revealed the increasing number of Men1−/− cells in mitosis. Cells were seeded at a density of 5 × 104, then counted on day 2, 4 and 6, and the cell growth curve revealed Men1 ablation increased the cell proliferation. In contrast, knockdown of cyclin B2 by shRNA diminished the number of cells in mitosis and reduced cell proliferation. Further, chromatin immunoprecipitation (ChIP) assay indicated that menin affected the histone modification of the promoter of Ccnb2 by reducing the level of histone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 acetylation but not affecting the level of histone H3 lysine 9 tri-methylation (H3K9me3) or histone H3 lysine 27 tri-methylation (H3K27me3). Our results suggest that menin may inhibit MEN1 insulinoma by suppressing cyclin B2 expression via histone modification.

Key words: insulinoma; cyclin B2; mitosis; cell proliferation; menin

收稿日期：2011-05-06　　录用日期：2011-06-16

通讯作者：吴婷　　E-mail: wuting78@yahoo.com.cn

引用本文：

吴婷, 黄小花. MEN1胰岛瘤中细胞周期蛋白cyclin B2高表达的作用和机制[J]. 生理学报 2011; 63 (6): 555-564.

WU Ting, HUANG Xiao-Hua. [The role and mechanism of high expression of cyclin B2 in MEN1 insulinoma.] [Article in Chinese]. Acta Physiol Sin 2011; 63 (6): 555-564 (in Chinese with English abstract).