结核分枝杆菌感染巨噬细胞lncRNA和mRNA表达谱的初步研究

发布时间：2018-04-12 23:07

  本文选题：基因芯片 + lncRNA　； 参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景结核分枝杆菌(Mycobacterium tuberculosis,MTB)是引起结核病的致病菌。MTB能通过多种免疫逃逸机制逃避巨噬细胞(macrophage,Mφ)的清除,从而长期寄生在Mφ内。长链非编码RNA(long noncoding RNA,lncRNA)广泛参与多种生物学进程并发挥关键作用,且可以作为多种疾病的诊断标志物和潜在的治疗靶标。但是lncRNA能否作为结核病的诊断标志物以及在MTB感染Mφ中的作用仍未清楚。研究目的通过基因芯片检测MTB感染Mφ后lncRNA和mRNA表达谱的变化,筛选有望用于结核病诊断的生物标志物;初步探讨了lncRNA—ENST00000360485在MTB感染Mφ中的作用,以期发现Mφ抵抗MTB感染的新机制。研究方法分别用MTB弱毒株H37Ra和强毒株H37Rv感染人原代巨噬细胞,使用lncRNA芯片(Arraystar Human LncRNA Microarray V3.0)检测被感染巨噬细胞lncRNA和mRNA表达谱,以未感染Mφ为对照;分别挑选6条lncRNA和6条mRNA,使用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)技术验证基因芯片结果的可重复性;使用生物信息学方法对差异表达的mRNA进行GO分析(Gene Ontology analysis,GO analysis)和 KEGG 通路分析(Kyoto Encyclopedia of Genes and Genomes(KEGG)biological pathway analysis);使用qRT-PCR检测32例健康志愿者和31例结核病患者的PBMC样本,筛选有望用于结核病诊断的生物标志物;使用siRNA抑制Mφ的lncRNA-ENST00000360485表达后,感染BCG,qRT-PCR检测其邻近基因和Mφ细胞因子TNF-α、IL-1β和IL-6的RNA表达水平变化,采用Western Blot检测Mφ自噬水平的变化,通过克隆形成实验(colony-forming unit,CFU)检测Mφ清除MTB的效果。结果基因芯片结果显示,H37Ra感染后,Mφ有972条lncRNA和2138条mRNA出现差异性表达,而H37Rv感染后,Mφ有1417条lncRNA和2478条mRNA出现差异性表达;所挑选的lncRNA和mRNA的表达趋势与基因芯片结果高度一致;GO和KEGG通路分析结果分别显示了 H37Ra和H37Rv感染相关的功能和代谢通路;qRT-PCR检测发现3个在健康对照和结核病人之间存在显著性差异表达的lncRNA(ENST00000360485,MIR3945HG V1 和MIR3945HG V2);ROC曲线分析结果显示:ENST00000360485、MIR3945HG V1、MIR3945HG V2的ROC曲线下面积(AUC)为0.798、0.925、0.956,敏感性分别是83.97%、90%、89.66%,特异性分别是71.88%、81.25%、90.63%;siRNA抑制ENST00000360485表达前后,ENST00000360485的上下游邻近基因(RAP1B和MDM1)和Mφ细胞因子TNF-α、IL-1β和IL-6的RNA表达水平没有变化,Mφ自噬通路的活化水平和清除MTB的能力也没有显著性差异。结论本研究发现,H37Rv和H37Ra诱导Mφ的lncRNA和mRNA表达谱具有显著性差异;生物信息学分析了差异表达的mRNA所富集的功能和代谢通路的差异;初步验证了3个lncRNA--ENST00000360485、MIR3945HG V1和MIR3945HG V2作为结核病诊断标志物的可行性;并对ENST00000360485功能的初步探讨,为后续深入研究奠定了基础。
[Abstract]:Background Mycobacterium tuberculosism MTB (Mycobacterium tuberculosisus MTB) is a pathogenic bacterium causing tuberculosis. MTB can escape the clearance of macrophage macrophage M 蠁 through a variety of immune escape mechanisms, so that it can be parasitized in M 蠁 for a long time.Long-chain noncoding RNA(long noncoding RNAs play a key role in many biological processes, and can be used as diagnostic markers and potential therapeutic targets for many diseases.However, whether lncRNA can be used as a diagnostic marker of tuberculosis and its role in M 蠁 infection of MTB remains unclear.Objective to detect the changes of lncRNA and mRNA expression profiles after MTB infection with M 蠁 by gene microarray, and to screen biomarkers that could be used in the diagnosis of tuberculosis, and to explore the role of lncRNA-ENST00000360485 in M 蠁 infection with MTB.The aim of this study was to find a new mechanism of M 蠁 resistance to MTB infection.Methods Human primary macrophages were infected with MTB attenuated H37Ra and virulent H37Rv, respectively. The lncRNA and mRNA expression profiles of infected macrophages were detected by lncRNA Human Human LncRNA Microarray V3.0.Six lncRNA and six mRNAs were selected, and the reproducibility of the results was verified by real-time fluorescence quantitative PCR(quantitative real-time real-time qRT-PCRR.Using bioinformatics method, go analysis of differentially expressed mRNA (go analysis) and KEGG pathway analysis of Genes and Genomes(KEGG)biological pathway analysis were performed. PBMC samples of 32 healthy volunteers and 31 tuberculosis patients were detected by qRT-PCR.After siRNA was used to inhibit the expression of M 蠁 lncRNA-ENST00000360485, the expression of adjacent genes and M 蠁 cytokines, TNF- 伪, IL-1 尾 and IL-6 were detected by RT-PCR, and M 蠁 autophagy was detected by Western Blot.Colony forming assay (CFU) was used to detect the MTB scavenging effect of M 蠁.Results there were 972 lncRNA and 2138 mRNA differentially expressed in M 蠁 after H37Ra infection, and 1417 lncRNA and 2478 mRNA in M 蠁 after H37Rv infection.The expression trend of selected lncRNA and mRNA was highly consistent with the results of gene chip analysis. The results of go and KEGG pathway analysis showed that H37Ra and H37Rv infection related function and metabolic pathway were detected by qRT-PCR in 3 healthy controls and TB patients, respectively.涔嬮棿瀛樺湪鏄捐憲鎬у樊寮傝〃杈剧殑lncRNA(ENST00000360485,MIR3945HG V1 鍜孧IR3945HG V2);ROC鏇茬嚎鍒嗘瀽缁撴灉鏄剧ず:ENST00000360485,MIR3945HG V1,MIR3945HG V2鐨凴OC鏇茬嚎涓嬮潰绉，

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