miR-99a-5p在结核分枝杆菌感染宿主巨噬细胞中的免疫调控作用

The role of miR-99a-5p in the immune regulation of host macrophages infected by Mycobacterium tuberculosis

目的:初步探讨结核分枝杆菌(Mycobacterium tuberculosis,MTB)感染THP-1细胞内特异miRNA在宿主抗结核免疫中的作用。方法:提取MTB感染及未感染THP-1细胞RNA,基于Illumina NovaSeq6000二代测序平台检测THP-1细胞内miRNA表达谱。采集4名健康受试者6 ml外周血并分离人原代巨噬细胞。应用实时荧光PCR(qRT-PCR)技术验证MTB感染THP-1细胞内特异miRNA并检测人原代巨噬细胞中靶标miRNA的表达水平。构建miRNA过表达载体,经慢病毒转染THP-1巨噬细胞获得稳定的过表达细胞株,使用qRT-PCR检测H37Rv感染的miRNA过表达细胞及对照空载体细胞中的肿瘤坏死因子α(TNF-α)及γ-干扰素(IFN-γ)表达水平。结果:从16个二代测序结果表达差异明显的miRNA分子中选取既往未作研究且差异最为明显的miR-99a-5p(P=0.021)作为研究靶基因。通过qRT-PCR在THP-1细胞及原代巨噬细胞中验证,发现miR-99a-5p在THP-1感染组和原代巨噬细胞内的表达量(分别为0.482±0.148和0.433±0.072)均明显低于未感染组(1.536±0.290和1.113±0.218),差异均有统计学意义(t=6.476,P<0.001;t=3.167,P=0.019)。MTB感染miR-99a-5p过表达细胞后24 h的菌落形成单位(CFU)[64.0×10^(4)(52.0×10^(4),87.0×10^(4))]明显高于MTB感染的对照空载体细胞内CFU[17.0×10^(4)(16.0×10^(4),24.0×10^(4))],差异有统计学意义(Z=-2.323,P=0.029)。与MTB感染的对照空载体细胞相比,MTB感染的miR-99a-5p过表达细胞内TNF-α及IFN-γ的mRNA相对表达水平分别由1.018±0.310和1.687±0.135下降至0.740±0.001和0.631±0.374,差异均有统计学意义(t=4.631,P=0.010;t=3.349,P=0.010)。结论:miR-99a-5p可抑制TNF-α及IFN-γ的释放,促进MTB在巨噬细胞内的生长。MTB感染后宿主miR-99a-5p的下调表达可能在宿主抗结核感染免疫中发挥重要作用。

Objective:To explore the role of specific microRNA in THP-1 cells mediated host defense against Mycobacterium tuberculosis(MTB)infection.Methods:Total RNA were extracted from THP-1 cells with or without MTB(H37Rv)infection,and then the miRNA expression profiles in THP-1 cells with or without MTB infection were identified using Illumina NovaSeq6000 next-generation sequencing platform.Four health controls were recruited and the human monocyte-derived macrophage(hMDM)were separated.Validation of differentially expressed miRNA between MTB infected and uninfected THP-1 cells,as well as that between MTB infected and uninfected hMDM,were performed by quantitative real-time PCR(qRT-PCR).The miRNA overexpression vector was constructed and transfected into THP-1 macrophages by lentivirus.The expression level of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ)and colony-forming unit(CFU)in H37Rv infected THP-1 cells which were transfected with miRNA overexpression vector or control empty vector was tested using qRT-PCR.Results:miR-99a-5p was selected as the study target gene(P=0.021)by not previously studied and most significantly different,from 16 miRNA molecules with significant expression differences in second-generation sequencing results.By qRT-PCR validation in THP-1 cells and primary macrophages,miR-99a-5p expression within THP-1 infected groups(0.482±0.148)and primary macrophages(0.433±0.072)were significantly lower than the uninfected group(1.536±0.290 and 1.113±0.218)(t=6.476,P<0.001;t=3.167,P=0.019).CFU results of the THP-1 cells infected by MTB showed that the intracellular bacteria amount of miR-99a-5p overexpression cells at 24 h after infection was 64.0×10^(4)(52.0×10^(4),87.0×10^(4)),which was significantly higher than that in the control cells(17.0×10^(4)(16.0×10^(4),24.0×10^(4)))(Z=-2.323,P=0.029).Furthermore,the expression levels of TNF-αand IFN-γin MTB infected miR-99a-5p over expression cells were 1.018±0.310 and 1.687±0.135,which were significantly lower than TNF-α(0.740±0.001)and IFN-γ(0.631±0.374)those in the MTB infected control cells(t=4.631,P=0.010;t=3.349,P=0.010).Conclusion:miR-99a-5p can inhibit the release of TNF-αand IFN-γ,promote the growth of MTB in macrophages.Therefore,the decreased miR-99a-5p expression after MTB infection may play a protective role in host innate immunity against MTB infection.