LncRNA GAS5靶向调控miR-144-3p对结核分枝杆菌感染的巨噬细胞凋亡和炎性反应的影响  

Impact of targeted regulation of miR-144-3p by LncRNA GAS5 on macrophage apoptosis and inflammatory response in Mycobacterium tuberculosis infected macrophages

作　　者：赵勇 梁丽丽[2] 江南[3] Zhao Yong;Liang Lili;Jiang Nan(Department of Tuberculosis,Shangqiu Chest Hospital,Henan Province,Shangqiu 476005,China;Department of Internal Medicine-Tuberculosis,Henan Provincial Thoracic Hospital,Zhengzhou 450008,China;Department of Tuberculosis Surgery,Henan Provincial Thoracic Hospital,Zhengzhou 450008,China)

机构地区：[1]河南省商丘市胸科医院结核科,商丘476005 [2]河南省胸科医院结核内科,郑州450008 [3]河南省胸科医院结核外科,郑州450008

出　　处：《中国防痨杂志》2023年第8期744-751,共8页Chinese Journal of Antituberculosis

基　　金：2019年河南省医学科技攻关计划(SB201903027)。

摘　　要：目的:探讨长链非编码RNA生长停滞特异性转录本5(LncRNA GAS5)靶向调控miR-144-3p对结核分枝杆菌(MTB)感染的巨噬细胞凋亡和炎性反应的影响。方法:qRT-PCR检测2019年3月31日至2022年3月31日在河南省商丘市胸科医院确诊的结核病患者75例、同期健康体检者75名血清中LncRNA GAS5、miR-144-3p水平;将巨噬细胞(160 nmol/L佛波酯诱导分化后的贴壁THP-1细胞)分为8组,即MTB组、MTB+pcDNA组、MTB+pcDNA-GAS5组、MTB+inhibitor NC组、MTB+miR-144-3p inhibitor组、MTB+pcDNA-GAS5+mimic NC组、MTB+pcDNA-GAS5+miR-144-3p mimic组,另取正常培养的巨噬细胞作为对照组(NC组)。检测巨噬细胞中LncRNA GAS5、miR-144-3p表达(qRT-PCR法)及细胞上清中炎性细胞因子水平(ELISA法);分别检测巨噬细胞增殖(CCK-8法)、凋亡(流式细胞术)及检测巨噬细胞中Bcl-2相关X蛋白(Bax)、B细胞淋巴瘤-2(Bcl-2)蛋白表达(Western blot)水平;验证LncRNA GAS5与miR-144-3p的关系(双荧光素酶报告基因实验)。结果:结核病患者血清中LncRNA GAS5表达量为0.24±0.02,低于健康体检者(1.00±0.00);结核病患者血清中miR-144-3p表达量为2.35±0.12,高于健康体检者(1.00±0.00),差异均有统计学意义(t值分别为329.090、97.428,P值均<0.001)。MTB组LncRNA GAS5表达量为0.22±0.02、细胞凋亡能力为(3.84±0.24)%、Bax蛋白表达量为0.22±0.02,均低于NC组[1.00±0.00、(9.79±0.23)%、1.21±0.12],miR-144-3p表达量(2.46±0.13)、炎性细胞因子水平[γ-干扰素(IFN-γ):(212.26±9.65)pg/ml;白细胞介素-6(IL-6):(123.35±5.12)pg/ml;肿瘤坏死因子α(TNF-α):(325.58±12.28)pg/ml]、细胞增殖能力(1.45±0.14)及Bcl-2蛋白表达量(1.53±0.15),均高于NC组[1.00±0.00、(35.58±1.23)pg/ml、(25.46±1.18)pg/ml、(51.12±2.03)pg/ml、0.86±0.07、0.56±0.05],差异均有统计学意义(q值分别为41.205、73.979、36.403、31.876、61.384、66.990、81.580、13.484、22.943,P值均<0.001)。MTB+pcDNA-GAS5组LncRNA GAS5表达量(0.86±0.07)、细胞凋亡能Objective:To investigate the impact of targeted regulation of miR-144-3p by long non-coding RNA growth arrest specific transcript 5(LncRNA GAS5)on macrophage apoptosis and inflammatory response in Mycobacterium tuberculosis(MTB)infected macrophages.Methods:Serum LncRNA GAS5 and miR-144-3p levels were detected by qRT-PCR in 75 patients diagnosed with tuberculosis and 75 healthy subjects who visited Shangqiu Chest Hospital,Henan Province from March 31,2019 to March 31,2022.Macrophages(160 nmol/L adherent THP-1 cells induced by phoboester)were divided into 8 groups:MTB group,MTB+pcDNA group,MTB+pcDNA-GAS5 group,MTB+inhibitor NC group,MTB+miR-144-3p inhibitor group,MTB+pcDNA-GAS5+mimic NC group,MTB+pcDNA-GAS5+miR-144-3p mimic group,normal culture macrophages were selected as control group(NC group).The expressions of LncRNA GAS5 and miR-144-3p in macrophages were detected by qRT-PCR and inflammatory cytokines in cell supernatant were detected by ELISA.Macrophage proliferation and apoptosis were detected by CCK-8 and flow cytometry respectively,and the expressions of Bcl-2 associated X protein(Bax)and B-cell lymphoma-2(Bcl-2)protein in macrophages were detected by Western Blot.The relationship between LncRNA GAS5 and miR-144-3p was verified(double luciferase reporter gene assay).Results:The average expression of LncRNA GAS5 in the serum of tuberculosis patients was 0.24±0.02,lower than that of the healthy group(1.00±0.00),and the expression of miR-144-3p was 2.35±0.12,higher than that of the healthy group(1.00±0.00),with statistical significance(t=329.090,97.428,all P<0.001).LncRNA GAS5 expression in MTB group was 0.22±0.02,apoptosis capacity was(3.84±0.24)%and Bax protein expression was 0.22±0.02,all lower than that in the NC group(1.00±0.00,(9.79±0.23)%,1.21±0.12).The expression of miR-144-3p(2.46±0.13),the inflammatory cytokine level(IFN-γ:(212.26±9.65)pg/ml;IL-6:(123.35±5.12)pg/ml;TNF-α:(325.58±12.28)pg/ml),the cell proliferation capacity(1.45±0.14)and Bcl-2 protein expression(1.53±0.15)were all

关 键 词：RNA探针 生长停滞特异性转录本5 分枝杆菌 结核 巨噬细胞 

分 类 号：R378[医药卫生—病原生物学]