lincRNA Cox2通过miR-129-5p/AMPK调控BCG感染的巨噬细胞糖酵解进程  

lincRNA Cox2 Regulates BCG-infected Macrophages Glycolysis by miR-129-5p/AMPK

作　　者：徐蕾 于嘉霖 刘莉 邓光存 吴晓玲 XU Lei;YU JiaLin;LIU Li;DENG GuangCun;WU XiaoLing(School of Life Sciences,Ningxia University/Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources in Western China,Yinchuan 750021)

机构地区：[1]宁夏大学生命科学学院/西部特色生物资源保护与利用教育部重点实验室,银川750021

出　　处：《中国农业科学》2024年第8期1606-1619,共14页Scientia Agricultura Sinica

基　　金：国家自然科学基金(32060160,32160162);宁夏自然科学基金重点项目(2023AAC02015)。

摘　　要：[目的]探究lincRNA Cox2对BCG(Bacillus Calmette-Guérin)感染的RAW264.7巨噬细胞糖酵解进程的调控作用,阐明Mtb与巨噬细胞之间的相互作用,为结核病的诊断和治疗提供新的靶点。[方法]利用小干扰RNA敲减lincRNA Cox2的表达,以及使用miR-129-5p mimics过表达载体,结合BCG感染,通过实时荧光定量PCR检测lincRNA Cox2、miR-129-5p和促炎因子IL-1β、TNF-α、IL-6的表达量;乳酸含量检测试剂盒检测乳酸(LD)的分泌情况;平板涂布法检测巨噬细胞菌载量情况;双荧光素酶报告基因系统验证lincRNA Cox2与miR-129-5p以及miR-129-5p与AMPK的互作关系;蛋白免疫印迹检测AMPK(AMP依赖蛋白激酶)及糖酵解途径中关键基因HK1(己糖激酶1)、PKM2(丙酮酸激酶2)和LDHA(乳酸脱氢酶)的表达变化。[结果]BCG感染12 h能够极显著上调RAW264.7巨噬细胞中lincRNA Cox2的表达(P=0.000013),与BCG组相比,siRNA+BCG组中AMPK(P=0.000771)、HK1(P=0.00323)、PKM2(P=0.000135)和LDHA(P=0.002532)的表达量以及乳酸的分泌量(P=0.020802)发生显著上调,而促炎因子IL-1β(P=0.000451)、TNF-α(P=0.000147)、IL-6(P=0.0001)的表达发生显著下调,菌载量试验表明siRNA+BCG组中的巨噬细胞菌载量显著下调(P=0.000127)。双荧光素酶报告基因系统表明lincRNA Cox2和miR-129-5p存在相互作用关系并以AMPK为靶基因。BCG感染RAW264.7巨噬细胞12 h后极显著下调miR-129-5p的表达(P=0.000156),与BCG组相比,miR-129-5p mimics+BCG组中AMPK(P=0.000262)、HK1(P=0.019524)、PKM2(P=0.001658)和LDHA(P=0.000887)表达量以及乳酸分泌量(P=0.044952)发生显著下调。[结论]lincRNA Cox2通过海绵吸附miR-129-5p并靶向AMPK,促进BCG感染的RAW264.7巨噬细胞糖酵解进程。【Objective】The aim of this study was to investigate the regulatory role of lincRNA Cox2 in the glycolysis of RAW264.7 macrophages infected by Bacillus Calmette-Guerin(BCG),and to elucidate the interaction between Mtb and macrophages,so as to provide a new target for the diagnosis and treatment of tuberculosis.【Method】RNA interference technique was used to knock down the expression of lincRNA Cox2,and miR-129-5p mimics were used to overexpress miR-129-5p.QPCR was performed to measure the lincRNA Cox2,miR-129-5p and proinflammatory cytokine(IL-1β,TNF-α,and IL-6)expression after BCG infection.The expression of Lactic Acid was detected by Lactic Acid assay kit.The bacterial load was measured bacterial load in BCG-infected macrophages.Dual luciferase reporter gene system validation experiments were carried out on lincRNA Cox2 and miR-129-5p,or miR-129-5p and AMPK relationships.The expression of AMPK(AMP activated protein kinase),HK1(Hexokinase 1),PKM2(pyruvate kinase M2),and LDHA(Lactate dehydrogenase A)were detected by Western blotting.【Result】The expression of lincRNA Cox2 was significantly upregulated(P=0.000013)after BCG infection in RAW264.7 macrophages for 12 h.Compared with the BCG group,the siRNA+BCG group had significantly upregulated the expression of AMPK(P=0.000771),HK1(P=0.00323),PKM2(P=0.000135),LDHA(P=0.002532),and the secretion of LD(P=0.020802),but the expression of IL-1β(P=0.000451),TNF-α(P=0.000147),IL-6(P=0.0001)was significantly reduced.The lincRNA Cox2 knockdown caused a significant reduce of bacterial load in BCG-infected macrophages(P=0.000127).Dual luciferase reporter gene system were performed to the co-localized of lincRNA Cox2 and miR-129-5p,and targeting AMPK.The expression of miR-129-5p was significantly reduced(P=0.000156)after BCG infection in RAW264.7 macrophages for 12 h.Compared with the BCG group,the miR-129-5p mimics+BCG group had significantly reduced the expression of AMPK(P=0.000262),HK1(P=0.019524),PKM2(P=0.001658),LDHA(P=0.000887),and the secretion of LD(P=0.04

关 键 词：lincRNA Cox2 miR-129-5p AMPK BCG 巨噬细胞 糖酵解进程 

分 类 号：S852.4[农业科学—基础兽医学]