Lnc RNA-GAS5对BCG感染小鼠巨噬细胞后细胞坏死的调控作用
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摘要
【背景】LncRNA-GAS5是由Gas5基因编码的功能性LncRNA分子,对细胞极化、细胞凋亡、坏死和自噬等多种生物学过程具有重要的调控作用。【目的】探讨LncRNA-GAS5对卡介苗(Bacillus Calmette-Guérin,BCG)诱导的小鼠巨噬细胞RAW264.7坏死的调控作用。【方法】构建LncRNA-GAS5的过表达及干扰载体,分别转染巨噬细胞RAW264.7后使用BCG感染,采用噻唑蓝比色法(MTT)检测细胞存活率,通过透射电镜观察细胞坏死形态,利用碘化丙啶(Propidine iodide,PI)单染法流式细胞仪检测细胞坏死率,通过实时荧光定量PCR和Western blot法研究坏死相关调控因子RIP1、RIP3和MLKL的表达水平。【结果】BCG感染巨噬细胞后,LncRNA-GAS5表达水平显著上调;同时,采用LncRNA-GAS5过表达载体单独转染或结合BCG感染后,巨噬细胞存活率均下降且细胞坏死率显著增加。同时坏死关键调控因子RIP1、RIP3和MLKL的mRNA及蛋白表达水平均显著上调(P<0.001),而GAS5干扰载体可有效抑制这一效果。【结论】LncRNA-GAS5通过上调坏死相关因子RIP1、RIP3及MLKL的表达促进BCG感染巨噬细胞后诱导的细胞坏死,研究结果为进一步探讨LncRNA-GAS5对BCG感染巨噬细胞后细胞坏死调控的分子机制奠定了基础。
[Background] LncRNA-GAS5, a functional LncRNA that is encoded by Gas5 gene, plays an important role in the regulation of cell polarization, apoptosis, necrosis and autophagy. [Objective] To investigate the regulatory function of LncRNA-GAS5 on necrosis of macrophage RAW 264.7 induced by Bacillus Calmette-Guérin(BCG) infection. [Methods] The overexpression plasmid and interference plasmid of LncRNA-GAS5 were constructed and transfected into macrophage RAW264.7. Furthermore,the both transfected macrophage were infected by BCG. The cell viability was detected by MTT assay. The morphological changes of necrosis were observed with transmission electron microscope and the necrosis rate was analyzed by flow cytometry. The expression of RIP1, RIP3 and MLKL in mRNA and protein level were determined by qRT-PCR and Western blot. [Results] The expression of LncRNA-GAS5 in macrophage RAW264.7 significantly increased after BCG infection. The cell viability decreased and cell necrosis rate increased significantly when macrophage was transfected with the LncRNA-GAS5 overexpression plasmid alone or combined with BCG infection. Meanwhile, the m RNA and protein expression levels of RIP1, RIP3 and MLKL were upregulated dramatically and this process can be inhibited by LncRNA-GAS5 interference plasmid. [Conclusion] LncRNA-GAS5 can promote necrosis of RAW264.7 infected with BCG by up-regulating necrosis associated factors such as RIP1, RIP3, and MLKL. The results lay foundation on further insight into the molecular mechanisms of macrophage necrosis induced by BCG infection.
[Background] LncRNA-GAS5, a functional LncRNA that is encoded by Gas5 gene, plays an important role in the regulation of cell polarization, apoptosis, necrosis and autophagy. [Objective] To investigate the regulatory function of LncRNA-GAS5 on necrosis of macrophage RAW 264.7 induced by Bacillus Calmette-Guérin(BCG) infection. [Methods] The overexpression plasmid and interference plasmid of LncRNA-GAS5 were constructed and transfected into macrophage RAW264.7. Furthermore,the both transfected macrophage were infected by BCG. The cell viability was detected by MTT assay. The morphological changes of necrosis were observed with transmission electron microscope and the necrosis rate was analyzed by flow cytometry. The expression of RIP1, RIP3 and MLKL in mRNA and protein level were determined by qRT-PCR and Western blot. [Results] The expression of LncRNA-GAS5 in macrophage RAW264.7 significantly increased after BCG infection. The cell viability decreased and cell necrosis rate increased significantly when macrophage was transfected with the LncRNA-GAS5 overexpression plasmid alone or combined with BCG infection. Meanwhile, the m RNA and protein expression levels of RIP1, RIP3 and MLKL were upregulated dramatically and this process can be inhibited by LncRNA-GAS5 interference plasmid. [Conclusion] LncRNA-GAS5 can promote necrosis of RAW264.7 infected with BCG by up-regulating necrosis associated factors such as RIP1, RIP3, and MLKL. The results lay foundation on further insight into the molecular mechanisms of macrophage necrosis induced by BCG infection.
引文
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[29]Zhao XM,Chen Z,Zhao JB,et al.Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis[J].Cell Death&Disease,2016,7(2):e2089
[30]Radonjic-Hoesli S,Wang XL,de Graauw E,et al.Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3(RIPK3)-mixed lineage kinase-like(MLKL)signaling pathway,which is counterregulated by autophagy[J].Journal of Allergy and Clinical Immunology,2017,140(6):1632-1642
[31]Wang HY,Sun LM,Su LJ,et al.Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3[J].Molecular Cell,2014,54(1):133-146
[32]Chen L,Yang WJ,Guo YJ,et al.Exosomal lncRNA GAS5regulates the apoptosis of macrophages and vascular endothelial cells in atherosclerosis[J].PLoS One,2017,12(9):e0185406
[33]Wang TH,Chan CW,Fang JY,et al.2-O-Methylmagnolol upregulates the long non-coding RNA,GAS5 and enhances apoptosis in skin cancer cells[J].Cell Death&Disease,2017,8(3):e2638
[2]Bionghi N,Padayatchi N,Master I,et al.Bedaquiline and linezolid for the operational treatment of multidrug-resistant and extensively drug-resistant tuberculosis in a high burden HIVsetting(BLIX Study)[J].American Journal of Respiratory and Critical Care Medicine,2017,195:A3090
[3]Zhang JM,Ma CJ,Zhao N,et al.Necrostatin-1 modulate the apoptosis induced by BCG in a macrophage cell line RAW264.7[J].Journal of Agricultural Biotechnology,2016,24(10):1552-1559(in Chinese)张嘉美,马臣杰,赵宁,等.Necrostatin-1对BCG感染后小鼠巨噬细胞RAW264.7凋亡的调控[J].农业生物技术学报,2016,24(10):1552-1559
[4]Dallenga T,Repnik U,Corleis B,et al.M.tuberculosis-induced necrosis of infected neutrophils promotes bacterial growth following phagocytosis by macrophages[J].Cell Host&Microbe,2017,22(4):519-530.e3
[5]Shiloh MU.Mechanisms of mycobacterial transmission:how does Mycobacterium tuberculosis enter and escape from the human host[J].Future Microbiology,2016,11(12):1503-1506
[6]Zhao X,Khan N,Gan H,et al.Bcl-x L mediates RIPK3-dependent necrosis in M.tuberculosis-infected macrophages[J].Mucosal Immunology,2017,10(6):1553-1568
[7]Wu XL,Deng GC,Hao XJ,et al.A caspase-dependent pathway is involved in Wnt/β-catenin signaling promoted apoptosis in Bacillus Calmette-Guerin infected RAW264.7 macrophages[J].International Journal of Molecular Sciences,2014,15(3):5045-5062
[8]Atianand MK,Hu WQ,Satpathy AT,et al.A long noncoding RNA lincRNA-EPS acts as a transcriptional brake to restrain inflammation[J].Cell,2016,165(7):1672-1685
[9]Kleaveland B,Shi CY,Stefano J,et al.A network of noncoding regulatory RNAs acts in the mammalian brain[J].Cell,2018.DOI:10.1016/j.cell.2018.05.022
[10]Schneider C,King RM,Philipson L.Genes specifically expressed at growth arrest of mammalian cells[J].Cell,1988,54(6):787-793
[11]Nakamura Y,Takahashi N,Kakegawa E,et al.The GAS5(growth arrest-specific transcript 5)gene fuses to BCL6 as a result of t(1;3)(q25;q27)in a patient with B-cell lymphoma[J].Cancer Genetics and Cytogenetics,2008,182(2):144-149
[12]Li F,Sun JG,Huang SL,et al.LncRNA GAS5 overexpression reverses LPS-induced inflammatory injury and apoptosis through up-regulating KLF2 expression in ATDC5 chondrocytes[J].Cellular Physiology and Biochemistry,2018,45(3):1241-1251
[13]Isin M,Ozgur E,Cetin G,et al.Investigation of circulating lncRNAs in B-cell neoplasms[J].Clinica Chimica Acta,2014,431:255-259
[14]Zhao QY,Wang XD,Sun XJ,et al.Light and transmission electron microscopic observation on the structure of zebrafish(Danio rerio)gill[J].Chinese Journal of Zoology,2018,53(1):92-98(in Chinese)赵巧雅,王新栋,孙雪婧,等.斑马鱼鳃的光镜和透射电镜观察[J].动物学杂志,2018,53(1):92-98
[15]Wu XL,Deng GC,Li M,et al.Wnt/β-Catenin signaling reduces Bacillus Calmette-Guerin-induced macrophage necrosis through a ROS-mediated PARP/AIF-dependent pathway[J].BMCImmunology,2015,16(1):16
[16]Castillo EF,Dekonenko A,Arko-Mensah J,et al.Autophagy protects against active tuberculosis by suppressing bacterial burden and inflammation[J].Proceedings of the National Academy of Sciences of the United States of America,2012,109(46):E3168-E3176
[17]Berghe TV,Linkermann A,Jouan-Lanhouet S,et al.Regulated necrosis:the expanding network of non-apoptotic cell death pathways[J].Nature Reviews Molecular Cell Biology,2014,15(2):135-147
[18]Pagán AJ,Yang CT,Cameron J,et al.Myeloid growth factors promote resistance to mycobacterial infection by curtailing granuloma necrosis through macrophage replenishment[J].Cell Host&Microbe,2015,18(1):15-26
[19]Han BW,Chen YQ.Non-codingRNAs in autophagy modulation[J].Chemistry of life,2014,34(4):434-441(in Chinese)韩博炜,陈月琴.非编码RNA与细胞自噬调控[J].生命的化学,2014,34(4):434-441
[20]Pickard MR,Williams GT.The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells[J].Oncotarget,2016,7(9):10104-10116
[21]Tao H,Zhang JG,Qin RH,et al.LncRNA GAS5 controls cardiac fibroblast activation and fibrosis by targeting miR-21 via PTEN/MMP-2 signaling pathway[J].Toxicology,2017,386:11-18
[22]Liu JY,Yao J,Li XM,et al.Pathogenic role of lncRNA-MALAT1in endothelial cell dysfunction in diabetes mellitus[J].Cell Death&Disease,2014,5(10):e1506
[23]Tran DDH,Kessler C,Niehus SE,et al.Myc target gene,long intergenic noncoding RNA,Linc00176 in hepatocellular carcinoma regulates cell cycle and cell survival by titrating tumor suppressor microRNAs[J].Oncogene,2018,37(1):75-85
[24]Zhang YY,Su SS,Zhao SB,et al.RIP1 autophosphorylation is promoted by mitochondrial ROS and is essential for RIP3recruitment into necrosome[J].Nature Communications,2017,8:14329
[25]Wallach D,Kang TB,Dillon CP,et al.Programmed necrosis in inflammation:toward identification of the effector molecules[J].Science,2016,352(6281):aaf2154
[26]Newton K,Manning G.Necroptosis and inflammation[J].Annual Review of Biochemistry,2016,85:743-763
[27]Zhang DW,Shao J,Lin J,et al.RIP3,an energy metabolism regulator that switches TNF-induced cell death from apoptosis to necrosis[J].Science,2009,325(5938):332-336
[28]Sun L,Wang H,Wang Z,et al.Mixed lineage kinase domain-like protein mediates necrosis signaling downstream of RIP3kinase[J].Cell,2012,148(1/2):213-227
[29]Zhao XM,Chen Z,Zhao JB,et al.Hsp90 modulates the stability of MLKL and is required for TNF-induced necroptosis[J].Cell Death&Disease,2016,7(2):e2089
[30]Radonjic-Hoesli S,Wang XL,de Graauw E,et al.Adhesion-induced eosinophil cytolysis requires the receptor-interacting protein kinase 3(RIPK3)-mixed lineage kinase-like(MLKL)signaling pathway,which is counterregulated by autophagy[J].Journal of Allergy and Clinical Immunology,2017,140(6):1632-1642
[31]Wang HY,Sun LM,Su LJ,et al.Mixed lineage kinase domain-like protein MLKL causes necrotic membrane disruption upon phosphorylation by RIP3[J].Molecular Cell,2014,54(1):133-146
[32]Chen L,Yang WJ,Guo YJ,et al.Exosomal lncRNA GAS5regulates the apoptosis of macrophages and vascular endothelial cells in atherosclerosis[J].PLoS One,2017,12(9):e0185406
[33]Wang TH,Chan CW,Fang JY,et al.2-O-Methylmagnolol upregulates the long non-coding RNA,GAS5 and enhances apoptosis in skin cancer cells[J].Cell Death&Disease,2017,8(3):e2638