长链非编码RNA Gm35082-202对布鲁氏菌引起的细胞焦亡的影响

doi:10.16431/j.cnki.1671-7236.2022.09.034

摘要/Abstract

摘要： 【目的】研究长链非编码RNA （lncRNA） Gm35082-202对布鲁氏菌侵染引起的细胞焦亡的影响，旨在为研究lncRNA参与布鲁氏菌引起的先天性免疫反应提供参考。【方法】在Ensemble数据库中查找并分析Gm35082-202在染色体上的位置、外显子等信息；采用RNAfold在线软件预测Gm35082-202的二级结构；通过KEGG、GO富集分析预测Gm35082-202功能；用牛种布鲁氏菌（S2308）以感染复数（MOI）为0.01侵染小鼠巨噬细胞（RAW264.7），在侵染不同时间点（0、4、8、12、24、36、48 h）收集细胞，用实时荧光定量PCR检测Gm35082-202的表达量；利用LncLocator网站预测Gm35082-202亚细胞定位，用实时荧光定量PCR检测Gm35082-202在细胞质、细胞核的表达；将3条Gm35082-202 siRNAs （siRNA1、siRNA2和siRNA3）、siRNA NC转染RAW264.7细胞，实时荧光定量PCR检测Gm35082-202的表达量，筛选干扰效率最高的siRNA进行后续试验。Gm35082-202-siRNA、Gm35082-202-siRNA-NC转染RAW264.7细胞24 h后，再用布鲁氏菌侵染，以PBS为空白对照组（PBS），只用布鲁氏菌侵染的细胞为侵染对照组（Bru）。侵染24 h后收集PBS组、Bru组、Gm35082-202-siRNA组（Bru-siRNA）、Gm35082-202-siRNA-NC组（Bru-NC）细胞，用实时荧光定量PCR和Western blotting分别检测各组细胞中NOD样受体蛋白3（NLRP3）、半胱氨酸天冬氨酸蛋白酶-1（Caspase-1）、Caspase-11、白细胞介素-1β（IL-1β）、IL-18的相对表达量，用ELISA法检测各组上清液中细胞因子IL-1β、IL-18分泌水平；通过菌落计数，比较各组布鲁氏菌的胞内生存能力。【结果】基因结构分析表明，Gm35082-202是大小为525 nt的lncRNA （Trianscript ID：ENSMUST00000208164.2），含有2个外显子，位于小鼠7号染色体（Chr7：100 324 295~100 326 967），其二级结构含有多个茎环及多分枝内部环结构；KEGG信号通路富集分析表明，Gm35082-202主要参与NOD样受体、钙调节以及细胞因子信号通路的调控；GO功能分析结果显示，Gm35082-202功能主要涉及DNA转录、线粒体组分以及金属离子结合。布鲁氏菌侵染RAW264.7细胞后，与0 h组相比，侵染4和8 h时Gm35082-202表达量显著增加（P<0.05），侵染12、24、36和48 h时表达量极显著增加（P<0.01）；亚细胞定位预测结果表明，Gm35082-202主要分布于细胞核（58.3%），其次是细胞质（35.1%），布鲁氏菌侵染RAW264.7细胞0 h时，Gm35082-202在细胞核中分布比例为63.4%，在侵染4、24和36 h Gm35082-202在细胞核中的分布减少，分别为24.5%、29.6%和30.4%。Gm35082-202-siRNA1、Gm35082-202-siRNA3均极显著降低Gm35082-202的表达量（P<0.01），Gm35082-202-siRNA1干扰效率最高。与Bru组相比，Bru-siRNA组中NLRP3、Caspase-1、Caspase-11、IL-1β和IL-18基因的表达量均极显著降低（P<0.01），NLRP3和Caspase-11蛋白的表达量显著降低（P<0.05），Caspase-1蛋白的表达量极显著降低（P<0.01）；细胞上清中IL-1β和IL-18分泌水平均极显著下降（P<0.01）；Bru-siRNA组胞内布鲁氏菌数显著升高（P<0.01）。【结论】布鲁氏菌通过上调Gm35082-202表达促进巨噬细胞焦亡，该结果可为进一步探究布鲁氏菌侵染时Gm35082-202参与调控宿主细胞焦亡的分子机制提供参考。

关键词: 布鲁氏菌; Gm35082-202; 细胞焦亡

Abstract: 【Objective】 This study was aimed to explore the effect of long non-coding RNA(lncRNA) Gm35082-20 on pyrolysis during Brucella infection, which laid a foundation for further research on the involvement of lncRNA in regulating immune response induced by Brucella.【Method】 The chromosome location and exon information of Gm35082-202 were searched and analyzed by Ensemble database, the secondary structure of Gm35082-202 was predicted though RNAfold online software, KEGG and GO enrichment analysis were used to investigate the function of Gm35082-202.RAW264.7 cells infected by Brucella at different time (0, 4, 8, 12, 24, 36 and 48 h) with MOI 0.01 were collected, and the expression of Gm35082-202 detected by Real-time quantitative PCR.The subcellular localization of Gm35082-202 was predicted by LncLocator website, and the expression of Gm35082-202 was detected by Real-time quantitative PCR in the nuclear and cytoplasmic.3 pieces siRNAs of Gm35082-202(siRNA1, siRNA2 and siRNA3) were designed and transfected to RAW264.7 cells, then the expression of Gm35082-202 was detected by Real-time quantitative PCR to screen siRNA with the highest interference efficiency for subsequent tests.After RAW264.7 cells were transinfected with Gm35082-202-siRNA and Gm35082-202-NC for 24 h, and then infected with Brucella.PBS was used as blank control group (PBS), and only Brucella infected cells were used as infection control group (Bru).Cells in PBS, Bru, Gm35082-202-siRNA (Bru-siRNA) and Gm35082-202-NC (Bru-NC) groups were collected, the mRNA and/or protein expressions of NLRP3, Caspase-1, Caspase-11, IL-1 and IL-18 were detected by Real-time quantitative PCR and Western blotting, respectively.The secretion levels of IL-1β and IL-18 in supernatant of each group were detect by ELISA.The intracellular viability of Brucella in each group was compared by colony count.【Result】 Gene structure analysis showed that Gm35082-202 was a lncRNA of 525 nt (Trianscript ID:ENSMUST00000208164.2), contained 2 exons, located in chromosome 7 of mouse(Chr7:100 324 295-100 326 967), its secondary structure contained multiple stem loops and multi-branched internal loop structures.KEGG signaling pathway enrichment analysis showed that Gm35082-202 mainly involved in regulating NOD-like receptor, calcium regulation and cytokine signaling pathway.GO function analysis showed that the function of Gm35082-202 mainly involved in DNA transcription, mitochondrial components and metal ion binding.After Brucella infected RAW264.7 cells, compared with 0 h group, the expression of Gm35082-202 was significantly increased at 4 and 8 h (P<0.05), and extremely significantly increased at 12, 24, 36 and 48 h (P<0.01).The prediction results of subcellular localization showed that Gm35082-202 was mainly distributed in nucleus (58%), followed by cytoplasm (35%).When RAW264.7 cells were infected by Brucella for 0 h, the distribution of Gm35082-202 in the nucleus was 63.4%, while the distribution of Gm35082-202 decreased to 24.5%, 29.6% and 30.4% at 4, 24 and 36 h, respectively.The expression of Gm35082-202 was extremely significantly reduced by Gm35082-202-siRNA1 and Gm35082-202-siRNA3 (P<0.01), and Gm35082-202-siRNA1 had the highest interference efficiency.Compared with Bru group, in Bru-siRNA group, the expressions of NLRP3, Caspase-1, Caspase-11, IL-1β and IL-10 genes were extremely significantly reduced(P<0.01), the expressions of NLRP3 and Caspase-11 protein were significantly decreased(P<0.05), whlie the expression of Caspase-1 protein was extremely significantly decreased(P<0.01), and the secretion of IL-1β and IL-18 in supernatant was extremely significantly decreased (P<0.01), and the intracellular Brucella count was increased significantly (P<0.05).【Conclusion】 Brucella promoted pyroapoptosis of macrophages by up-regulating Gm35082-202 expression, which could provide references for further exploring the molecular mechanism of Gm35082-202 in regulating pyroapoptosis of host cells during Brucella infection.

Key words: Brucella; Gm35082-202; pyroptosis

中图分类号:

S852.61⁺4

邓兴梅, 曹树珠, 郭嘉, 朱德馨, 赵天艺, 柴迎锦, 张伟, 史超, 贾思锋, 张辉. 长链非编码RNA Gm35082-202对布鲁氏菌引起的细胞焦亡的影响[J]. 中国畜牧兽医, 2022, 49(9): 3599-3609.

DENG Xingmei, CAO Shuzhu, GUO Jia, ZHU Dexin, ZHAO Tianyi, CHAI Yingjin, ZHANG Wei, SHI Chao, JIA Sifeng, ZHANG Hui. Effect of Long Non-coding RNA Gm35082-202 on Cell Pyrodecay Induced by Brucella[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(9): 3599-3609.

参考文献

[1] 权伍荣, 杨咏洁.布鲁氏菌逃逸宿主的抗感染免疫机制[J].微生物学报, 2016, 56(5):747-752. QUAN W R, YANG Y J.Anti-infection immune mechanism of Brucella escaping the host[J].Acta Microbiologica Sinica, 2016, 56(5):747-752.(in Chinese)
[2] DORNELES E, TEIXEIRA-CARVALHA, MSS A, et al.Immune response triggered by Brucella abortus following infection or vaccination[J].Vaccine, 2015, 33(31):3659-3666.
[3] MARIM F M, MIRIAM M, FRANC C, et al.The role of NLRP3 and AIM2 in inflammasome activation during Brucella abortus infection[J].Seminars in Immunopathology, 2017, 39(2):215-223.
[4] MARTINON F, BURNS K, TSCHOPP J.The inflammasome:A molecular platform triggering activation of inflammatory caspases and processing of proIL-beta[J].Molecular Cell, 2002, 10(2):417-426.
[5] LISTON A, MASTERS S L.Homeostasis-altering molecular processes as mechanisms of inflammasome activation[J].Nature Reviews Immunology, 2017, 17(3):208-214.
[6] BROZ P.Recognition of intracellular bacteria by inflammasomes[J].Microbiology Spectrum, 2019, 7(2):BAI-0003.
[7] LACEY C A, MITCHELL W J, DADELAHI A S, et al.Caspase-1 and Caspase-11 mediate pyroptosis, inflammation, and control of Brucella joint infection[J].Infection and Immunity, 2018, 86(9):361-379.
[8] SHI J, ZHAO Y, WANG K, et al.Cleavage of GSDMD by inflammatory caspases determines pyroptotic cell death[J].Nature, 2015, 526(7575):660-665.
[9] ELLING R, CHAN J, FITZGERALD K A.Emerging role of long noncoding RNAs as regulators of innate immune cell development and inflammatory gene expression[J].European Journal of Immunology, 2016, 46(3):504-512.
[10] GAO J, CHEN X, WEI P, et al.Regulation of pyroptosis in cardiovascular pathologies:Role of noncoding RNAs[J].Molecular Therapy-Nucleic Acids, 2021, 25(9):220-236.
[11] ZHANG C, GONG Y, LI N, et al.Long non-coding RNA Kcnq1ot1 promotes sC5b-9-induced podocyte pyroptosis by inhibiting miR-486a-3p and upregulating NLRP3[J].American Journal of Physiology-cell Physiology, 2020, 320(3):C355-C364.
[12] LING H, LI Q, DUAN Z P, et al.lncRNA GAS5 inhibits miR-579-3p to activate SIRT1/PGC-1α/Nrf2 signaling pathway to reduce cell pyroptosis in sepsis-associated renal injury[J].American Journal of Physiology-cell Physiology, 2021, 321(1):C117-C133.
[13] SUZUKI T, FRANCHI L, TOMA C, et al.Differential regulation of Caspase-1 activation, pyroptosis, and autophagy via Ipaf and ASC in shigella-infected macrophages[J].PLoS Pathogens, 2007, 3(8):e111.
[14] ZAMBONI D S, KOBAYASHI K S, KOHLSDORF T, et al.The birc1e cytosolic pattern-recognition receptor contributes to the detection and control of legionella pneumophila infection[J].Nature in Immunology, 2006, 7(3):318-325.
[15] CERQUEIRA D M, GOMES M, SILVA A, et al.Guanylate-binding protein 5 licenses Caspase-11 for gasdermin-D mediated host resistance to Brucella abortus infection[J].PLoS Pathogens, 2018, 14(12):e1007519.
[16] KHAN M, HARMS J S, LIU Y, et al.Brucella suppress STING expression via miR-24 to enhance infection[J].PLoS Pathogens, 2020, 16(10):e1009020.
[17] ZHANG P, CAO L, ZHOU R, et al.The lncRNA Neat1 promotes activation of inflammasomes in macrophages[J].Nature Communications, 2019, 10(1):1495-1512.
[18] VANDE WALLE L, LAMKANFI M.Pyroptosis[J].Current Biology, 2016, 13(11):568-572.
[19] YU Z W, ZHANG J, LI X, et al.A new research hot spot:The role of NLRP3 inflammasome activation, a key step in pyroptosis, in diabetes and diabetic complications[J]. Life Sciences, 2020, 240:117138.
[20] MIAO E, LEAF I, TREUTING P, et al.Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria[J].Nature Immunology, 2010, 11(12):1136-1142.
[21] WEN Y, CHEN H, LUO F, et al.Roles of long noncoding RNAs in bacterial infection[J].Life Sciences, 2020, 263:118579.
[22] GOMEZ J A, WAPINSKI O L, YANG Y W, et al.The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-gamma locus[J].Cell, 2013, 152(4):743-754.
[23] DENG X, GUO J, SUN Z, et al.Brucella-induced downregulation of lncRNA Gm28309 triggers macrophages inflammatory response through the miR-3068-5p/NF-κB pathway[J].Frontiers in Immunology, 2020, 11:581517.
[24] HU W, ALVAR-DOMINGUEZ J, LOSISH H F.Regulation of mammalian cell differentiation by long non-coding RNAs[J].EMBO Reports, 2012, 13(11):971-983.