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

doi:10.19982/j.issn.1000-6621.20230105

摘要/Abstract

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(160nmol/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)、细胞凋亡能力[(7.62±0.17)%]及Bax蛋白表达量(0.94±0.08),均高于MTB+pcDNA组[0.23±0.01、(3.82±0.21)%、0.23±0.01];miR-144-3p表达量(1.35±0.10)、炎性细胞因子水平[IFN-γ:(96.63±4.17)pg/ml;IL-6:(41.93±2.19)pg/ml;TNF-α:(118.85±6.03)pg/ml]、细胞增殖能力(0.96±0.08)及Bcl-2蛋白表达量(0.81±0.07),均明显低于MTB+pcDNA组[2.48±0.14、(210.63±9.78)pg/ml、(125.52±4.86)pg/ml、(327.73±10.15)pg/ml、1.44±0.13、1.54±0.14],差异均有统计学意义(q值分别为33.281、47.247、26.107、24.671、57.204、61.448、62.087、10.970、17.266,P值均<0.001)。MTB+miR-144-3p inhibitor组细胞凋亡能力[(7.51±0.19)%]及Bax蛋白表达量(0.89±0.08),均高于MTB+inhibitor NC组[(3.86±0.22)%、0.24±0.02];miR-144-3p表达量(1.21±0.09)、炎性细胞因子水平[IFN-γ:(103.36±5.11)pg/ml;IL-6:(39.95±1.62)pg/ml;TNF-α:(120.26±6.67)pg/ml]、细胞增殖能力(0.92±0.08)及Bcl-2蛋白表达量(0.78±0.07),均低于MTB+inhibitor NC组[2.47±0.12、(214.45±10.03)pg/ml、(126.05±4.77)pg/ml、(326.69±8.33)pg/ml、1.46±0.12、1.54±0.12],差异均有统计学意义(q值分别为45.382、23.901、27.509、58.921、61.029、61.359、12.341、17.976,P值均<0.001)。结论: 过表达LncRNA GAS5可能通过靶向下调miR-144-3p抑制MTB感染的巨噬细胞炎性反应,并诱导细胞凋亡。

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

Abstract:

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 higher than the NC group (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), the differences were all statistically significant (q=41.205, 73.979, 36.403, 31.876, 61.384, 66.990, 81.580, 13.484, 22.943, all P<0.001). In MTB+pcDNA-GAS5 group, the expression of LncRNA GAS5 was 0.86±0.07, the apoptosis capacity was (7.62±0.17) % and the expression of Bax protein was 0.94±0.08, higher than that in the MTB+pcDNA group (0.23±0.01, (3.82±0.21) %, 0.23±0.01); the expression of miR-144-3p (1.35±0.10), and the inflammatory cytokine level (IFN-γ: (96.63±4.17) pg/ml; IL-6: (41.93±2.19) pg/ml; TNF-α: (118.85±6.03) pg/ml), cell proliferation capacity (0.96±0.08) and Bcl-2 protein expression (0.81±0.07) were all lower than that in the MTB+pcDNA group (2.48±0.14, (210.63±9.78) pg/ml, (125.52±4.86) pg/ml, (327.73±10.15) pg/ml, 1.44±0.13, 1.54±0.14), the differences were all statistically significant (q=33.281, 47.247, 26.107, 24.671, 57.204, 61.448, 62.087, 10.970, 17.266, all P<0.001). The apoptotic capacity of MTB+miR-144-3p inhibitor group was (7.51±0.19) % and the expression of Bax protein was 0.89±0.08, which were higher than that of MTB+inhibitor NC group ((3.86±0.22) %, 0.24±0.02). The expression of miR-144-3p (1.21±0.09), the inflammatory cytokine level (IFN-γ: (103.36±5.11) pg/ml; IL-6: (39.95±1.62) pg/ml; TNF-α: (120.26±6.67) pg/ml), cell proliferation capacity (0.92±0.08) and Bcl-2 protein expression (0.78±0.07) were all lower than that in the MTB+inhibitor NC group (2.47±0.12, (214.45±10.03) pg/ml, (126.05±4.77) pg/ml, (326.69±8.33) pg/ml, 1.46±0.12, 1.54±0.12), and all differences were statistically significant (q=45.382, 23.901, 27.509, 58.921, 61.029, 61.359, 12.341, 17.976, all P<0.001). Conclusion: Overexpression of LncRNA GAS5 might inhibit the inflammatory response of macrophages infected with MTB and induce apoptosis through targeted down-regulation of miR-144-3p.

Key words: RNA probe, Growth arrest specific transcript 5, Mycobacterium tuberculosis, Macrophages

中图分类号:

R378

赵勇, 梁丽丽, 江南. 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[J]. 中国防痨杂志, 2023, 45(8): 744-751. doi: 10.19982/j.issn.1000-6621.20230105

Zhao Yong, Liang Lili, Jiang Nan. Impact of targeted regulation of miR-144-3p by LncRNA GAS5 on macrophage apoptosis and inflammatory response in Mycobacterium tuberculosis infected macrophages[J]. Chinese Journal of Antituberculosis, 2023, 45(8): 744-751. doi: 10.19982/j.issn.1000-6621.20230105

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