HDAC2对急性缺血性卒中患者中性粒细胞TBC蛋白家族成员的表观调控

Epigenetic regulation of HDAC2 on TBC protein family members of neutrophils in patients with ischemic stroke

目的研究急性缺血性卒中(acute ischemic stroke,AIS)后介导中性粒细胞脱颗粒和吞噬功能的分子以及组蛋白去乙酰化酶2(histone deacetylase 2,HDAC2)的表观调控机制。方法从首都医科大学宣武医院急诊选取3例前循环缺血发病6 h内未接受溶栓和血管内治疗的60~80岁男性患者,另选3例性别、年龄匹配的健康对照者,采用密度梯度离心的方法分离外周血中性粒细胞,进行RNA测序(RNA sequencing,RNA-seq),分析AIS患者与健康对照组的转录组差异;另外,通过染色质免疫共沉淀测序(chromatin immunoprecipitation high-throughput sequencing,ChIP-seq)分析中性粒细胞中与HDAC2结合的基因。结果(1)RNA-seq显示,与健康对照组相比,AIS后13个TBC1D家族分子转录水平变化显著,其中TBC蛋白家族成员-TBC1D10A、TBC1D31、TBC1D5共3个分子的转录水平显著下调,TBC1D10B、TBC1D10C、TBC1D17、TBC1D2、TBC1D22A、TBC1D26、TBC1D29、TBC1D3H、TBC1D8、TBC1D9B共10个分子转录水平显著上调。(2)ChIP-seq分析显示,AIS患者的HDAC2结合的TBC1D家族基因差异性表达,对HDAC2结合的差异基因启动子进行甲基化分析,发现这些TBC1D家族分子存在高度甲基化和中度甲基化修饰,受表观遗传学乙酰化和甲基化双重调节。结论TBC1D家族分子可能是调节急性缺血性卒中后中性粒细胞脱颗粒和吞噬的潜在靶点,HDAC2与该家族的基因组可能有广泛的结合。

Objective To study the molecular mechanism that mediating neutrophil degranulation and phagocytosis after acute ischemic stroke(AIS)and the epigenetic regulatory mechanism of histone deacetylase 2(HDAC2).Methods Three 60-80 years old male patients from the Department of Emergency,Xuanwu Hospital,Capital Medical University who did not receive thrombolysis or endovascular treatment within 6 h after anterior circulation ischemia onset and three healthy gender and age-matched controls were included in this study.The peripheral blood neutrophils was isolated with density gradient centrifugation method,and the transcriptome difference between AIS patients and healthy controls was analyzed with RNA sequencing(RNA-seq).In addition,HDAC2 binding genes in neutrophils were analyzed with chromatin immunoprecipitation high-throughput sequencing(ChIP-seq).Results(1)RNA-seq results indicated that compared with the healthy control group,the transcription level of 13 TBC1D family molecules changed significantly after AIS,among which TBC protein family members TBC1D10A,TBC1D31,and TBC1D5 were significantly down-regulated,and TBC1D10B,TBC1D10C,TBC1D17,TBC1D2,TBC1D22A,TBC1D26,TBC1D29,TBC1D3H,TBC1D8,and TBC1D9B were significantly up-regulated.(2)ChIP-seq analysis indicated that HDAC2 binding genes of TBC1D family in AIS patients were differentially expressed.Methylation analysis of HDAC2 binding promoters showed that these TBC1D family molecules were modified by hypermethylation and moderate methylation,which were regulated by acetylation and methylation modification.Conclusion This study showed that TBC1D family molecules may be potential targets for regulating neutrophil degranulation and phagocytosis after ischemic stroke,and HDAC2 may have extensive binding sites in the genome of this family.