心肌祖细胞中Tbx5启动子区的组蛋白乙酰化调控机制研究

Study of regulatory mechanism of histone acetylation in Tbx5 promoter region of cardiac progenitor cells

目的研究心肌祖细胞中Tbx5启动子区p300和CBP介导的组蛋白乙酰化修饰对Tbx5表达的调控及其修饰位点,探讨Tbx5的组蛋白乙酰化修饰调控网络。方法体外培养胚胎心肌祖细胞,分为对照组、二甲基亚砜(DMSO)组和姜黄素组,对照组不给予额外干预,DMSO组给予DMSO干预,姜黄素组给予30 mmol/L姜黄素干预。同时,采用慢病毒载体转染胚胎心肌细胞48 h,分为对照组、GFP组和p300-RNAi组,对照组不转染,GFP组采用GFP慢病毒载体转染,p300-RNAi组采用p300-RNAi慢病毒载体转染。Western blot方法检测各组的组蛋白H3的乙酰化水平;实时荧光定量逆转录PCR检测各组Tbx5和p300 mRNA水平的表达;染色质免疫共沉淀(CHIP)-实时荧光定量逆转录PCR检测Tbx5启动子区组蛋白H3不同位点H3K4、H3K9和H3K27的乙酰化水平及Tbxt5启动子结合的p300和CBP水平。结果姜黄素组Tbx5 mRNA相对表达水平及H3ac水平较对照组和DMSO对照组明显降低,差异有统计学意义(P<0.05)。p300-RNAi组p300 mRNA相对表达水平、H3ac水平较GFP组和对照组明显降低,差异有统计学意义(P<0.05);3组Tbx5 mRNA相对表达水平比较,差异无统计学意义(P>0.05)。与GFP组和对照组比较,p300-RNAi组H3ac、H3K4ac、H3K27ac水平降低,H3K9ac水平升高,差异有统计学意义(P<0.05)。p300、CBP均与Tbx5启动子区域相结合。与GFP组和对照组比较,p300-RNAi组p300结合水平降低,CBP结合水平升高,差异有统计学意义(P<0.05)。结论Tbx5启动子区组蛋白乙酰化修饰受p300和CBP的双重调控,p300-RNAi介导的H3K4和H3K27低乙酰化对Tbx5表达的抑制作用,可被CBP介导的H3K9高乙酰化代偿,保证Tbx5的正确表达。

Objective To study the regulation of histone acetylation in Tbx5 promoter region mediated by p300 and CBP in cardiac progenitor cells and their modification sites,and to explore the histone acetylation regulatory network of Tbx5.Methods Embryonic myocardial progenitor cells were cultured in vitro and divided into the control group,DMSO group and curcumin group.The control group was not given the additional intervention.The DMSO group was given the DMSO intervention.The curcumin group was given 30mmol/L curcumin intervention.At the same time,the lentiviral vector was adopted to transfect to embryo myocardial cells for 48 h.They were divided into the control group,GFP group and p300-RNAi group.The control group had no transfection,the GFP group adopted the GFP lentiviral vector transfection,and the p300-RNAi group adopted the p300-RNAi lentiviral vector transfection.Western blot was used to detect the acetylation level of histone H3 in each group.The mRNA expression levels of Tbx5 and p300 were detected by RT-PCR.CHIP-RT-PCR was used to detect the acetylation levels of histone H3 different sites of H3K4,H3K9 and H3K27 in the Tbx5 promoter region and the levels of p300 and CBP combined with Tbxt5 promoter region.Results The Tbx5 mRNA relative expression level and H3ac level in the curcumin group were significantly reduced compared with the control group and DMSO group,and the differences were statistically significant(P<0.05).The p300 mRNA relative expression level and H3ac level in the p300-RNAi group were significantly decreased compared with GFP group and control group,and the differences were statistically significant(P<0.05).The Tbx5 mRNA relative expression level had no statistical difference among the three groups(P>0.05).Compared with the GFP group and control group,the H3ac,H3K4ac and H3K27ac levels in the p300-RNAi group were decreased,the H3K9ac level was increased,and the differences were statistically significant(P<0.05).p300 and CBP were combined with the Tbx5 promoter region.Compared with the GFP group and control group,the p300 combination level in the p300-RNAi group was decreased,the CBP combination level was increased,and the differences were statistically significant(P<0.05).Conclusion The histone acetylation modification in the Tbx5 promoter region is regulated by both p300 and CBP.The low acetylation of H3K4 and H3K27 mediated by p300-RNAi has the inhibiting effect on the Tbx5 expression,and could be compensated by CBP mediated H3K9 high acetylation for ensuring the Tbx5 correct expression.