乳酸调控小鼠巨噬细胞M2型极化的关键靶点分子筛选

Screening of key target molecules for lactic acid regulation of M2-type polarization of mouse macrophages

目的:乳酸堆积是肿瘤微环境(TME)呈酸性的主要原因之一,巨噬细胞是TME中的重要免疫细胞。本研究筛选乳酸调控小鼠腹腔巨噬细胞M2型极化的关键靶点分子,为巨噬细胞相关的肿瘤免疫治疗提供理论依据。方法:提取小鼠源腹腔巨噬细胞,通过流式细胞术检测F4/80+及CD11b+细胞表达量;将巨噬细胞分为2组,分别用0 mmol/L(对照)、20 mmol/L乳酸的培养基处理24 h后收集细胞,qPCR法检测巨噬细胞IL-10、Arg-1、VEGF、HIF-1等基因的相对表达量;利用转录组测序技术筛选对照组和20 mmol/L乳酸组的差异表达基因(|log2(fold change)|>1),对差异表达基因进行GO及KEGG分析,并利用Cytoscape软件将筛选出的差异表达基因构建互作网络图,根据基因互作节点数筛选出Tpt1及Malat1等关键调控基因。分别将Tpt1、Malat1两个关键基因的siRNA转染至巨噬细胞,抑制两个基因的表达,利用qPCR法检测转染后巨噬细胞CD11c、Arg-1、VEGF等基因的相对表达量。结果:获得的小鼠腹腔细胞中F4/80+及CD11b+细胞的表达量均大于90%,表明成功获得高纯度的小鼠腹腔来源巨噬细胞。与对照组相比,经20 mmol/L乳酸处理后,Arg-1、HIF-1、IL-10和VEGF表达量增加,表明乳酸能促进巨噬细胞的M2型极化。转录组学研究结果发现,20 mmol/L乳酸处理24 h后使巨噬细胞的6 295个基因表达发生变化,差异表达基因主要参与细菌防御反应、胞质基因翻译、核糖体生物合成、胎盘发育等生物过程;涉及的信号通路包括MAPK、PI3KAkt、NOD样受体等。筛选出关键上调差异表达基因Malat1、Tpt1、Fadd、Gm5900、Gipr等。Malat1或Tpt1表达被抑制后,20 mmol/L乳酸均能上调巨噬细胞中CD11c表达,抑制VEGF、Arg-1表达。结论:乳酸可能通过上调Malat1及Tpt1表达进而促进巨噬细胞的M2型极化。

Objective:Lactic acid accumulation is one of the main causes of acidic tumor microenvironment(TME),and macrophages are important cells in TME.In this study,we screened the key target molecules in the regulation of M2-type polarization of mouse peritoneal macrophages by lactic acid,and provided theoretical basis for macrophage-related tumor immunotherapy.Methods:Peritoneal macrophages were extracted from mice and the expression levels of F4/80+and CD11b+cells were detected by flow cytometry.Macrophages were divided into 2 groups,and treated with 0 mmol/L(control)and 20 mmol/L lactic acid medium for 24 h,respectively.The cells were collected,and the relative expression levels of IL-10,Arg-1,VEGF,HIF-1 and other genes in macrophages were detected by qPCR.Transcriptome sequencing technology was used to screen the differentially expressed genes(|log2(fold change)|>1)in the control group and the 20 mmol/L lactic acid group.GO and KEGG analysis were performed on the differentially expressed genes,and Cytoscape software was used to construct the interaction network diagram of the differentially expressed genes.Key regulatory genes such as Tpt1 and Malat1 were screened according to the number of gene interaction nodes.siRNA of two key genes Tpt1 and Malat1 were transfected into macrophages respectively to inhibit the expressions of the two genes.The relative expression levels of CD11c,Arg-1 and VEGF genes in the transfected macrophages were detected by qPCR method.Results:The expression levels of F4/80+and CD11b+cells in mouse peritoneal cells were both greater than 90%,indicating that we have successfully obtained high-purity mouse peritoneal macrophages.Compared with control group,the expression levels of Arg-1,HIF-1,IL-10 and VEGF increased after 20 mmol/L lactic acid treatment,suggesting that lactic acid can promote M2-type polarization of macrophages.Transcriptome study showed that 6295 genes in macrophages were changed after 20 mmol/L lactic acid treatment for 24 h,and the differentially expressed genes were mainly involved in bacterial defense response,cytoplasmic gene translation,ribosomal biosynthesis,placental development and other biological processes.The involved signaling pathways include MAPK,PI 3 K-Akt,NOD-like receptor,etc..Key up-regulated differentially expressed genes,such as Malat 1,Tpt 1,Fadd,Gm 5900 and Gipr,were screened.After Malat1 or Tpt1 expressions were inhibited,20 mmol/L lactic acid could up-regulate the expression of CD 11 c in macrophages and inhibit the expressions of VEGF and Arg-1.Conclusion:Lactic acid may promote M 2-type polarization of macrophages by up-regulating Malat 1 and Tpt 1 expressions.