iPS细胞向内皮分化过程中Wnt信号对组蛋白去乙酰化酶5的调控  

Wnt-mediated HDAC5 Regulation during Endothelial Differentiation of iPS Cells

作　　者：唐琦凯 王雨晴 张菲雨 伍浩鹏 张婉怡 李涛 TANG Qi-Kai;WANG Yu-Qing;ZHANG Fei-Yu;WU Hao-Peng;ZHANG Wan-Yi;LI Tao(Department of Medical Laboratory,School of Medicine,Hunan Normal University,Changsha 410013,China;Grade 2021 Medical Laboratory Technology Class,School of Medicine,Hunan Normal University,Changsha 410013,China;Grade 2021 Excellent Doctor Class,School of Medicine,Hunan Normal University,Changsha 410013,China)

机构地区：[1]湖南师范大学医学院检验系,长沙410013 [2]湖南师范大学医学院,长沙410013

出　　处：《中国生物化学与分子生物学报》2024年第6期838-847,共10页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金(No.81870201);湖南省自然科学基金(No.2022JJ30411);湖南省教育厅科学研究项目(No.23A0074);2023年度湖南省大学生创新创业训练计划一般项目(No.S202310542209);湖南师范大学思政示范课程和医学院教改课题资助。

摘　　要：HDAC(histone deacetylase)是一类可对蛋白质进行去乙酰化修饰的表观修饰酶,通过改变细胞核内组蛋白乙酰化状态,调控启动子活化水平,从而影响下游基因的表达水平,但其在内皮分化过程中的表达变化尚不清楚。本研究利用3阶段法诱导hiPSCs向内皮细胞定向分化,利用qRT-PCR检测Ⅰ类HDAC(HDAC 1、2)、Ⅱ类HDAC(HDAC 4、5)基因的表达变化。研究发现,HDAC5分子在内皮分化中有着显著的表达变化,在中胚层分化阶段下调90%(P<0.01),在血管前体阶段上调至3.7倍(P<0.01),继而在内皮晚期分化阶段下调70%(P<0.01)。免疫印迹结果进一步证实,HDAC5蛋白在内皮分化过程中存在阶段性表达变化。机制研究中显示,HDAC5中胚层分化阶段的变化受Wnt信号调控。通过CHIR99021处理以及Wnt3a质粒过表达,激动Wnt信号通路,会引起HDAC5下调。通过IWP-2抑制Wnt信号通路,会促进HDAC5表达恢复。此外研究发现,HDAC5主要定位于细胞核,IWP-2恢复HDAC5表达,但大部分滞留在胞浆。进一步研究显示,中胚层分化阶段HDAC5下调与中胚层分化标志物BraT的表达启动相关。通过HDAC抑制剂BML210处理发现,其可以促进早期中胚层分化,干扰血管前体细胞的内皮分化,增强内皮晚期阶段分化。总体而言,内皮诱导分化过程中,HDAC5有着显著的阶段性表达改变。Wnt信号激活是调控HDAC5在中胚层分化阶段下调的主要机制。HDAC(histone deacetylase)is a class of epigenetic modifying enzymes that can deacetylate proteins by altering the acetylation status of histones in the nucleus,regulating promoter activation levels,and thereby affecting downstream gene expression.However,expression changes of HDACs during endothelial differentiation are still unclear.This study used a three-stage method to induce human induced pluripotent stem cells(hiPSCs)to differentiate into endothelial cells,and qRT-PCR was used to detect the expression changes of class I HDAC(HDAC 1,2)and class II HDAC(HDAC 4,5)genes.It was found that HDAC 5 exhibits significant expression changes during endothelial differentiation.It is downregulated by 90%during the mesodermal differentiation stage(P<0.01),upregulated by 3.7-fold during the vascular precursor stage(P<0.01),and subsequently downregulated by 70%during the late stage of endothelial differentiation(P<0.01).Immunoblotting experiments further confirmed that HDAC5 undergoes periodic expression changes during endothelial differentiation.Mechanistic studies have shown that HDAC5 downregulation during the differentiation stage of the mesoderm is mediated by Wnt signaling.CHIR99021 treatment and overexpression of Wnt3a can activate the Wnt signaling pathway,leading to HDAC5 downregulation.Inhibiting the Wnt signaling pathway through IWP-2 promotes the recovery of HDAC5 expression.In addition,it was found that HDAC5 is mainly localized in the nucleus,and IWP-2 restores HDAC5 expression,but it remains in the cytoplasm.Further research suggests that downregulation of HDAC5 during mesodermal differentiation may contribute to the expression of the mesodermal marker BraT.Treatment with the HDAC inhibitor BML210 can promote early mesodermal differentiation,interfere with endothelial differentiation of vascular precursor cells,and enhance late-stage endothelial differentiation.In conclusion,HDAC5 displays a stage-specific expression during endothelial differentiation,and Wnt signaling activation is the main mechanism regulat

关 键 词：人诱导多能干细胞 内皮细胞 组蛋白去乙酰化酶 表达调控 WNT信号 

分 类 号：Q593[生物学—生物化学]