不同毒性结核分枝杆菌感染巨噬细胞后基因组DNA甲基化水平的变化

Changes and differences of DNA methylation in human macrophages infected with virulent and avirulent Mycobacterium tuberculosis

目的比较不同毒性MTB感染巨噬细胞后基因组DNA甲基化水平的变化。方法以对数生长期的MTB实验室标准株H37Rv和H37Ra分别感染经佛波酯（50 ng/ml）诱导分化的人单核巨噬细胞系（THP－1）作为实验组：THP－1/Ra组和THP－1/Rv组，同时设置未感染MTB的对照组。分别提取3份细胞样本的基因组DNA，采用简化甲基化测序（reduced representation bisulfite sequencing，RRBS）方法进行甲基化测序。用生物信息学分析方法比较各样本DNA整体甲基化水平，重点分析样本间差异性甲基化区域（differentially methylated region，DMR），对DMR位于启动子区的相关基因进行功能注释、基因本体论（GO）聚类及京都基因与基因组百科全书（KEGG）通路分析，最终通过实时荧光定量PCR方法验证部分基因的表达。两样本间差异分析采用t检验。结果THP－1/Rv组、THP－1/Ra组以及对照组中甲基化C碱基以CG类型为主，占全部甲基化C碱基类型（CG、CHG、CHH）的98%以上；3组样本中基因启动子区的所有C碱基平均甲基化水平分别为7.72%、7.38%和7.58%；CpG岛（CGI）所有C碱基平均甲基化水平分别为9.90%、9.57%和9.80%。与THP－1/Ra组相比，THP－1/Rv组基因组DNA上定位到58个差异DMR，其区域平均长度为152 bp。对DMR位于启动子区的相关基因进行GO聚类及KEGG通路分析，发现6个高度富集于13条通路的基因，其中DNA损伤诱导转录子4（DDIT4）和转录激活蛋白1（AP－1）在THP－1/Ra组和THP－1/Rv组的表达具有显著差异。结论MTB感染巨噬细胞系后细胞基因组未出现整体甲基化水平的增加或降低，而部分区域出现甲基化差异，且不同毒性MTB感染组表现出区域甲基化水平的差异。此外，DDIT4和AP－1可能是2个新的与MTB毒性相关的基因。

Objective To profile DNA methylation and compare differentially methylated region （DMR） of macrophages infected with virulent and avirulent strains of Mycobacterium tuberculosis （MTB）. Methods PMA（50 ng/ml） treated THP-1 macrophages were left uninfected or infected with the virulent H37Rv（THP-1/Rv） or avirulent H37Ra（THP-1/Ra）. The genomic DNA was then extracted and DNA methylation was profiled via Reduced Representation Bisulfite Sequencing （RRBS）. The DNA methylation pattern and DMRs between the tested samples were indentified. DMRs-assoeiated genes were annotated, clustered by Gene Ontology （GO） , and the pathways analyzed by Kyoto Encyclopedia of Genes and Genomes （KEGG）. mRNA expression of several DMR-associated genes were verified by Q-PCR, and Student＇s t-test was used to analyze the differential expression between either 2 samples. Results The mostly DNA methylated C in 3 samples was in CG, accounting for 98% of all types of methylated C（CG, CGH, CHH）. Besides, the average DNA methylation level in THP-1/Rv, THP-1/Ra and THP-1/NC were 7.72%, 7.38% and 7.58% in the promoter, and 9.90%, 9. 57% and 9.80% in the CpG island （CGI）, respectively. We identified 58 DMRs between H37Rv and H37Ra infected THP-1 macrophages with an average length of 152 bp. Six DMRs-associated genes were enriched. Among them, AP-1 and DDIT4 were differentially expressed in cells infected with H37Rv and H37Ra. Conclusions Infection with MTB is not correlated with a large-scale gain or loss in DNA methylation in host cells. AP-1 and DDIT4 might be novel virulence-related genes regulated by DNA methylation of their corresponding promoter regions.