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。
【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.
作者
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
DENG Xingmei;CAO Shuzhu;GUO Jia;ZHU Dexin;ZHAO Tianyi;CHAI Yingjin;ZHANG Wei;SHI Chao;JIA Sifeng;ZHANG Hui(College of Animal Science and Technology,Shihezi University,Shihezi 832000,China;Key Laboratory of Control and Prevention of Animal Disease,Xinjiang Production&Construction Corps,Shihezi 832000,China;State International Joint Research Center for Animal Health Breeding,Shihezi University,Shihezi 832000,China;Qilu Animal Health Products Co.,Ltd.,Shihezi 832000,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第9期3599-3609,共11页
China Animal Husbandry & Veterinary Medicine
基金
重点领域科技攻关计划(2021AB012)
国家自然科学基金(31860691)。