摘要目的 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.

abstractsObjective 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-associated 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.