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

doi:10.3864/j.issn.0578-1752.2024.08.014

摘要/Abstract

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。

关键词: lincRNA Cox2, miR-129-5p, AMPK, BCG, 巨噬细胞, 糖酵解进程

Abstract:

【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=0.044952). 【Conclusion】 lincRNA Cox2 promoted BCG-infected RAW264.7 macrophages glycolysis process by sponging miR-129-5p and targeting AMPK.

Key words: lincRNA Cox2, miR-129-5p, AMPK, BCG, macrophages, glycolysis

徐蕾, 于嘉霖, 刘莉, 邓光存, 吴晓玲. lincRNA Cox2通过miR-129-5p/AMPK调控BCG感染的巨噬细胞糖酵解进程[J]. 中国农业科学, 2024, 57(8): 1606-1619.

XU Lei, YU JiaLin, LIU Li, DENG GuangCun, WU XiaoLing. lincRNA Cox2 Regulates BCG-infected Macrophages Glycolysis by miR-129-5p/AMPK[J]. Scientia Agricultura Sinica, 2024, 57(8): 1606-1619.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2024.08.014

https://www.chinaagrisci.com/CN/Y2024/V57/I8/1606

图/表 9

表1

图1

图2

图3

图4

图5

图6

图7

图8

参考文献 37

[1]	Global tuberculosis report 2022. Geneva: World Health Organization, 2022.
[2]	于嘉霖, 徐雅楠, 韩璐, 马沁梅, 吴晓玲, 邓光存. 脂肪酸结合蛋白4对BCG诱导巨噬细胞自噬的调控作用. 畜牧兽医学报, 2020, 51(9): 2265-2274.
	YU J L, XU Y N, HAN L, MA Q M, WU X L, DENG G C. Role of fatty acid binding protein 4 in regulating macrophage autophagy induced by BCG infection. Chinese Journal of Animal and Veterinary Sciences, 2020, 51(9): 2265-2274. (in Chinese)
[3]	毕秀欣, 韩鹏宇, 马吉雪, 李发. 新冠肺炎疫情对全球结核病防治的影响. 口岸卫生控制, 2022, 27(2): 48-51.
	BI X X, HAN P Y, MA J X, LI F. Impact of COVID-19 on global tuberculosis prevention. Port Health Control, 2022, 27(2): 48-51. (in Chinese)
[4]	PEPPERELL C S. Evolution of tuberculosis pathogenesis. Annual Review of Microbiology, 2022, 76: 661-680. doi: 10.1146/micro.2022.76.issue-1
[5]	YANG J S, REN B, YANG G, WANG H Y, CHEN G Y, YOU L, ZHANG T P, ZHAO Y P. The enhancement of glycolysis regulates pancreatic cancer metastasis. Cellular and Molecular Life Sciences, 2020, 77(2): 305-321. doi: 10.1007/s00018-019-03278-z pmid: 31432232
[6]	MENDONCA L E, PERNET E, KHAN N, SANZ J, KAUFMANN E, DOWNEY J, GRANT A, ORLOVA M, SCHURR E, KRAWCZYK C, JONES R G, BARREIRO L B, DIVANGAHI M. Human alveolar macrophage metabolism is compromised during Mycobacterium tuberculosis infection. Frontiers in Immunology, 2022, 13: 1044592. doi: 10.3389/fimmu.2022.1044592
[7]	GLEESON L E, O’LEARY S M, RYAN D, MCLAUGHLIN A M, SHEEDY F J, KEANE J. Cigarette smoking impairs the bioenergetic immune response to Mycobacterium tuberculosis infection. American Journal of Respiratory Cell and Molecular Biology, 2018, 59(5): 572-579. doi: 10.1165/rcmb.2018-0162OC
[8]	HUANG L, NAZAROVA E V, TAN S M, LIU Y C, RUSSELL D G. Growth of Mycobacterium tuberculosis in vivo segregates with host macrophage metabolism and ontogeny. The Journal of Experimental Medicine, 2018, 215(4): 1135-1152. doi: 10.1084/jem.20172020
[9]	HACKETT E E, CHARLES-MESSANCE H, O’LEARY S M, GLEESON L E, MUÑOZ-WOLF N, CASE S, WEDDERBURN A, JOHNSTON D G W, WILLIAMS M A, SMYTH A, OUIMET M, MOORE K J, LAVELLE E C, CORR S C, GORDON S V, KEANE J, SHEEDY F J. Mycobacterium tuberculosis limits host glycolysis and IL-1β by restriction of PFK-M via microRNA-21. Cell Reports, 2020, 30(1): 124-136.e4. doi: 10.1016/j.celrep.2019.12.015
[10]	BRIDGES M C, DAULAGALA A C, KOURTIDIS A. LNCcation: lncRNA localization and function. The Journal of Cell Biology, 2021, 220(2): e202009045. doi: 10.1083/jcb.202009045
[11]	于志瑞, 张旭, 牛莎莎, 邓光存, 吴晓玲. LncRNA NR₀03508通过海绵吸附miR-483-3p并靶向MLKL调控BCG感染小鼠巨噬细胞坏死. 畜牧兽医学报, 2022, 53(9): 3149-3159.
	YU Z R, ZHANG X, NIU S S, DENG G C, WU X L. LncRNA NR₀03508 regulates BCG-infected mouse macrophages necrosis by the sponge adsorption of miR-483-3p and targeting MLKL. Acta Veterinaria et Zootechnica Sinica, 2022, 53(9): 3149-3159. (in Chinese)
[12]	YAO Q L, XIE Y, XU D D, QU Z L, WU J, ZHOU Y Y, WEI Y Y, XIONG H, ZHANG X L. Lnc-EST12, which is negatively regulated by mycobacterial EST12, suppresses antimycobacterial innate immunity through its interaction with FUBP3. Cellular & Molecular Immunology, 2022, 19(8): 883-897.
[13]	LIU L, YU Z R, MA Q M, YU J L, GONG Z Q, DENG G C, WU X L. LncRNA NR_003508 suppresses Mycobacterium tuberculosis- induced programmed necrosis via sponging miR-346-3p to regulate RIPK1. International Journal of Molecular Sciences, 2023, 24(9): 8016. doi: 10.3390/ijms24098016
[14]	ROBINSON E K, WORTHINGTON A, POSCABLO D, SHAPLEIGH B, SALIH M M, HALASZ H, SENINGE L, MOSQUEIRA B, SMALIY V, FORSBERG E C, CARPENTER S. lincRNA-Cox2 functions to regulate inflammation in alveolar macrophages during acute lung injury. Journal of Immunology, 2022, 208(8): 1886-1900. doi: 10.4049/jimmunol.2100743 pmid: 35365562
[15]	LIU Q, JIANG J W, FU Y, LIU T, YU Y, ZHANG X F. MiR-129-5p functions as a tumor suppressor in gastric cancer progression through targeting ADAM9. Biomedicine & Pharmacotherapy, 2018, 105: 420-427. doi: 10.1016/j.biopha.2018.05.105
[16]	GAO B, WANG L J, ZHANG N, HAN M M, ZHANG Y B, LIU H C, SUN D L, XIAO X L, LIU Y F. MiR-129-5p inhibits clear cell renal cell carcinoma cell proliferation, migration and invasion by targeting SPN. Cancer Cell International, 2021, 21(1): 263. doi: 10.1186/s12935-021-01820-3 pmid: 34001147
[17]	SEN K, PATI R, JHA A, MISHRA G P, PRUSTY S, CHAUDHARY S, SWETALIKA S, PODDER S, SEN A, SWAIN M, NANDA R K, RAGHAV S K. NCoR1 controls immune tolerance in conventional dendritic cells by fine-tuning glycolysis and fatty acid oxidation. Redox Biology, 2023, 59: 102575. doi: 10.1016/j.redox.2022.102575
[18]	DE JESUS A, KEYHANI-NEJAD F, PUSEC C M, GOODMAN L, GEIER J A, STOOLMAN J S, STANCZYK P J, NGUYEN T, XU K, SURESH K V, CHEN Y H, RODRIGUEZ A E, SHAPIRO J S, CHANG H C, CHEN C L, SHAH K P, BEN-SAHRA I, LAYDEN B T, CHANDEL N S, WEINBERG S E, ARDEHALI H. Hexokinase 1 cellular localization regulates the metabolic fate of glucose. Molecular Cell, 2022, 82(7): 1261-1277.e9. doi: 10.1016/j.molcel.2022.02.028 pmid: 35305311
[19]	WIESE E K, HITOSUGI S, LOA S T, SREEDHAR A, ANDRES-BECK L G, KURMI K, PANG Y P, KARNITZ L M, GONSALVES W I, HITOSUGI T. Enzymatic activation of pyruvate kinase increases cytosolic oxaloacetate to inhibit the Warburg effect. Nature Metabolism, 2021, 3(7): 954-968. doi: 10.1038/s42255-021-00424-5 pmid: 34226744
[20]	DING J, KARP J E, EMADI A. Elevated lactate dehydrogenase (LDH) can be a marker of immune suppression in cancer: Interplay between hematologic and solid neoplastic clones and their microenvironments. Cancer Biomarkers, 2017, 19(4): 353-363. doi: 10.3233/CBM-160336 pmid: 28582845
[21]	MURALEEDHARAN R, DASGUPTA B. AMPK in the brain: its roles in glucose and neural metabolism. The FEBS Journal, 2022, 289(8): 2247-2262. doi: 10.1111/febs.v289.8
[22]	杨舟, 林书典, 詹宇威, 肖璐, 符克英, 黄小蝶. LncRNA MIR22HG 通过海绵吸附miR-22-5p对类风湿关节炎成纤维样滑膜细胞增殖、凋亡和炎性反应的影响. 安徽医科大学学报, 2023, 58(3): 405-412.
	YANG Z, LIN S D, ZHAN Y W, XIAO L, FU K Y, HUANG X D. Effects of lncRNA MIR22HG on proliferation, apoptosis and inflammatory response of rheumatoid arthritis fibroblast-like synoviocytes by sponge adsorption of miR-22-5p. Acta Universitatis Medicinalis Anhui, 2023, 58(3): 405-412. (in Chinese)
[23]	BOSEDASGUPTA S, PIETERS J. Inflammatory stimuli reprogram macrophage phagocytosis to macropinocytosis for the rapid elimination of pathogens. PLoS Pathogens, 2014, 10(1): e1003879. doi: 10.1371/journal.ppat.1003879
[24]	HOWARD N C, KHADER S A. Immunometabolism during Mycobacterium tuberculosis infection. Trends in Microbiology, 2020, 28(10): 832-850. doi: 10.1016/j.tim.2020.04.010
[25]	RANSOHOFF J D, WEI Y N, KHAVARI P A. The functions and unique features of long intergenic non-coding RNA. Nature Reviews Molecular Cell Biology, 2018, 19(3): 143-157. doi: 10.1038/nrm.2017.104 pmid: 29138516
[26]	NELSON B R, MAKAREWICH C A, ANDERSON D M, WINDERS B R, TROUPES C D, WU F F, REESE A L, MCANALLY J R, CHEN X W, KAVALALI E T, CANNON S C, HOUSER S R, BASSEL- DUBY R, OLSON E N. A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle. Science, 2016, 351(6270): 271-275. doi: 10.1126/science.aad4076 pmid: 26816378
[27]	WEI L, LIU K, JIA Q Z, ZHANG H, BIE Q L, ZHANG B. The roles of host noncoding RNAs in Mycobacterium tuberculosis infection. Frontiers in Immunology, 2021, 12: 664787. doi: 10.3389/fimmu.2021.664787
[28]	冉宏标, 赵丽玲, 王会, 柴志欣, 王吉坤, 王嘉博, 武志娟, 钟金城. LncFAM200B对牦牛肌内前体脂肪细胞脂质沉积的影响. 中国农业科学, 2022, 55(13): 2654-2666. doi: 10.3864/j.issn.0578-1752.2022.13.014.
	RAN H B, ZHAO L L, WANG H, CHAI Z X, WANG J K, WANG J B, WU Z J, ZHONG J C. Effects of lnc FAM200B on the lipid deposition in intramuscular preadipocytes of yak. Scientia Agricultura Sinica, 2022, 55(13): 2654-2666. doi: 10.3864/j.issn.0578-1752.2022.13.014. (in Chinese)
[29]	禹保军, 邓占钊, 辛国省, 蔡正云, 顾亚玲, 张娟. 静原鸡肌肉组织肌苷酸特异性沉积相关LNC_003828-gga-miR-107-3p-MINPP1的关联分析. 中国农业科学, 2021, 54(19): 4229-4242. doi: 10.3864/j.issn.0578-1752.2021.19.017.
	YU B J, DENG Z Z, XIN G S, CAI Z Y, GU Y L, ZHANG J. Correlation analysis of inosine monophosphate specific deposition related LNC_003828-gga-miR-107-3P-MINPP1 in Jingyuan chicken muscle tissue. Scientia Agricultura Sinica, 2021, 54(19): 4229-4242. doi: 10.3864/j.issn.0578-1752.2021.19.017. (in Chinese)
[30]	JIANG F, LOU J, ZHENG X M, YANG X Y. LncRNA MIAT regulates autophagy and apoptosis of macrophage infected by Mycobacterium tuberculosis through the miR-665/ULK1 signaling axis. Molecular Immunology, 2021, 139: 42-49. doi: 10.1016/j.molimm.2021.07.023
[31]	LI D Y, GAO C Y, ZHAO L, ZHANG Y M. Inflammatory response is modulated by lincRNACox2 via the NF-κB pathway in macrophages infected by Mycobacterium tuberculosis. Molecular Medicine Reports, 2020, 21(6): 2513-2521.
[32]	HE Y N, WANG Y T, JIA X B, LI Y X, YANG Y, PAN L F, ZHAO R, HAN Y, WANG F, GUAN X Y, HOU T Z. Glycolytic reprogramming controls periodontitis-associated macrophage pyroptosis via AMPK/ SIRT1/NF-κB signaling pathway. International Immunopharmacology, 2023, 119: 110192. doi: 10.1016/j.intimp.2023.110192
[33]	CHIN W Y, HE C Y, CHOW T W, YU Q Y, LAI L C, MIAW S C. Adenylate kinase 4 promotes inflammatory gene expression via Hif1α and AMPK in macrophages. Frontiers in Immunology, 2021, 12: 630318. doi: 10.3389/fimmu.2021.630318
[34]	GAUTHIER T, YAO C, DOWDY T, JIN W W, LIM Y J, PATIÑO L C, LIU N, OHLEMACHER S I, BYNUM A, KAZMI R, BEWLEY C A, MITROVIC M, MARTIN D, MORELL R J, ECKHAUS M, LARION M, TUSSIWAND R, O’SHEA J J, CHEN W J. TGF-β uncouples glycolysis and inflammation in macrophages and controls survival during sepsis. Science Signaling, 2023, 16(797): eade0385. doi: 10.1126/scisignal.ade0385
[35]	Ó MAOLDOMHNAIGH C, COX D J, PHELAN J J, MITERMITE M, MURPHY D M, LEISCHING G, THONG L, O’LEARY S M, GOGAN K M, MCQUAID K, COLEMAN A M, GORDON S V, BASDEO S A, KEANE J. Lactate alters metabolism in human macrophages and improves their ability to kill Mycobacterium tuberculosis. Frontiers in Immunology, 2021, 12: 663695. doi: 10.3389/fimmu.2021.663695
[36]	方舒. LncRNA-Cox2对BCG诱导RAW264.7细胞自噬的调控作用[D]. 西宁: 宁夏大学, 2021.
	FANG S. Regulation of LncRINA-Cox2 on autophagy of RAW264.7 induced by Bacillus Calmette-Guérin[D]. Xining: Ningxia University, 2021. (in Chinese)
[37]	XU Y N, YU J L, MA C J, GONG Z Q, WU X L, DENG G C. Impact of knockdown LincRNA-Cox2 on apoptosis of macrophage infected with Bacillus Calmette-Guérin. Molecular Immunology, 2021, 130: 85-95. doi: 10.1016/j.molimm.2020.11.008

引物名称 Primer	引物序列Sequence（5'→3'）
lincRNA Cox2	F: AAGGAAGCTTGGCGTTGTGA
lincRNA Cox2	R: GAGAGGTGAGGAGTCTTATG
miR-129-5p	F: ACACTCCAGCTGGCCTTTTTCGCCAGACC CGAA
miR-129-5p	R: TGGTGTCGTGGAGTCG
IL-1β	F: CAGGCAGGCAGTATCACTCATTG
IL-1β	R: CGTCACACACCAGCAGGTTATC
TNF-α	F: TAGCCCATGTTGTAGCAAACC
TNF-α	R: ATGAGGTACAGGCCCTCTGAT
IL-6	F: GACAGCCACTCACCTCTTCAG
IL-6	R: CATCCATCTTTTTCAGCCATC
β-actin	F: GTGCTATGTTGCTCTAGACTTCG
β-actin	R: ATGCCACAGGATTCCATACC
U6	F: CTCGCTTCGGCAGCACA
U6	R: AACGCTTCACGAATTTGCGT