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

doi:10.19982/j.issn.1000-6621.20230105

Abstract

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

CLC Number:

R378

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

Figures/Tables 7

References 22

[1]	Fu Y, Shen J, Liu F, et al. Andrographolide Suppresses Pyroptosis in Mycobacterium tuberculosis-Infected Macrophages via the microRNA-155/Nrf2 Axis. Oxid Med Cell Longev, 2022, 2022:1885066. doi:10.1155/2022/1885066. doi: 10.1155/2022/1885066
[2]	Marks GB, Nguyen NV, Nguyen PTB, et al. Community-wide screening for tuberculosis in a high-prevalence setting. N Engl J Med, 2019, 381(14):1347-1357. doi:10.1056/NEJMoa1902129. doi: 10.1056/NEJMoa1902129 URL
[3]	刘冬梅, 彭少君, 韩晓群, 等. STAT1在PPARγ活化后抑制结核分枝杆菌感染巨噬细胞炎症反应中的作用. 免疫学杂志, 2022, 38(9):768-774. doi:10.13431/j.cnki.immunol.j.20220107. doi: 10.13431/j.cnki.immunol.j.20220107
[4]	Li Y, Sun L, Liu J, et al. Down-regulation of GAS5 has diagnostic value for tuberculosis and regulates the inflammatory response in Mycobacterium tuberculosis infected THP-1 cells. Tuberculosis (Edinb), 2022, 132:102141. doi:10.1016/j.tube.2021.102141. doi: 10.1016/j.tube.2021.102141 URL
[5]	Looney M, Lorenc R, Halushka MK, et al. Key macrophage responses to infection with Mycobacterium tuberculosis are co-regulated by microRNAs and DNA methylation. Front Immunol, 2021, 12:685237. doi:10.3389/fimmu.2021.685237. doi: 10.3389/fimmu.2021.685237
[6]	Guo L, Zhou L, Gao Q, et al. MicroRNA-144-3p inhibits autophagy activation and enhances Bacillus Calmette-Guérin infection by targeting ATG4a in RAW264.7 macrophage cells. PLoS One, 2017, 12(6):e0179772. doi:10.1371/journal.pone.0179772. doi: 10.1371/journal.pone.0179772
[7]	Ji Q, Qiao X, Liu Y, et al. Expression of long-chain nonco-ding RNA GAS 5 in osteoarthritis and its effect on apoptosis and autophagy of osteoarthritis chondrocytes. Histol Histopathol, 2021, 36(4):475-484. doi:10.14670/HH-18-312. doi: 10.14670/HH-18-312
[8]	中华人民共和国国家卫生和计划生育委员会. WS 288—2017 肺结核诊断. 2017-11-09.
[9]	杨盛娅, 孙亚萍, 刘立宾, 等. miR-150调控PDCD4对结核分枝杆菌H37Rv感染THP-1巨噬细胞的影响. 中国免疫学杂志, 2021, 37(5):547-552,563. doi:10.3969/j.issn.1000-484X.2021.05.007. doi: 10.3969/j.issn.1000-484X.2021.05.007
[10]	Sun W, Zhang X, He X, et al. Long non-coding RNA SNHG 16 silencing inhibits proliferation and inflammation in Mycobacterium tuberculosis-infected macrophages by targeting miR-140-5p expression. Infect Genet Evol, 2022, 103:105325. doi:10.1016/j.meegid.2022.105325. doi: 10.1016/j.meegid.2022.105325 URL
[11]	Pattanaik KP, Ganguli G, Naik SK, et al. Mycobacterium tuberculosis EsxL induces TNF-α secretion through activation of TLR2 dependent MAPK and NF-κB pathways. Mol Immunol, 2021, 130:133-141. doi:10.1016/j.molimm.2020.11.020. doi: 10.1016/j.molimm.2020.11.020 pmid: 33419561
[12]	宋蔷, 周康仕, 李典芬, 等. 结核分枝杆菌诱导的人肺泡巨噬细胞对IFN-γ反应性的改变及机制研究. 医学理论与实践, 2017, 30(12):1730-1732. doi:10.19381/j.issn.1001-7585.2017.12.006. doi: 10.19381/j.issn.1001-7585.2017.12.006
[13]	Sousa J, Cá B, Maceiras AR, et al. Mycobacterium tuberculosis associated with severe tuberculosis evades cytosolic surveillance systems and modulates IL-1β production. Nat Commun, 2020, 11(1):1949-1962. doi:10.1038/s41467-020-15832-6. doi: 10.1038/s41467-020-15832-6
[14]	Yao Q, Xie Y, Xu D, et al. Lnc-EST12, which is negatively regulated by mycobacterial EST12, suppresses antimycobacterial innate immunity through its interaction with FUBP3. Cell Mol Immunol, 2022, 19(8):883-897. doi:10.1038/s41423-022-00878-x. doi: 10.1038/s41423-022-00878-x
[15]	唐才环, 薛芳, 陈乙云, 等. 肺炎支原体肺炎患儿血清中lncRNA MALAT1和lncRNA GAS5表达与病情严重程度及预后的相关性分析. 中国病原生物学杂志, 2022, 17(10):1199-1203. doi:10.13350/j.cjpb.221018. doi: 10.13350/j.cjpb.221018
[16]	张丛敏, 刘颖, 刘静, 等. HBV宫内感染孕妇血清LncRNA GAS5、ZEB1的表达及其临床意义. 中国病案, 2021, 22(9):97-101. doi:10.3969/j.issn.1672-2566.2021.09.034. doi: 10.3969/j.issn.1672-2566.2021.09.034
[17]	Zeng Z, Lan Y, Chen Y, et al. LncRNA GAS5 suppresses inflammatory responses by inhibiting HMGB1 release via miR-155-5p/SIRT1 axis in sepsis. Eur J Pharmacol, 2023, 942(1):175520. doi:10.1016/j.ejphar.2023.175520. doi: 10.1016/j.ejphar.2023.175520
[18]	Yang L, Zhang X, Liu X. Long noncoding RNA GAS5 protects against Mycoplasma pneumoniae pneumonia by regulating the microRNA2223p/TIMP3 axis. Mol Med Rep, 2021, 23(5):380. doi:10.3892/mmr.2021.12019. doi: 10.3892/mmr.2021.12019
[19]	Zhao Y, Yuan D, Zhu D, et al. LncRNA-MSC-AS1 inhibits the ovarian cancer progression by targeting miR-425-5p. J Ovarian Res, 2021, 14(1):109-119. doi:10.1186/s13048-021-00857-2. doi: 10.1186/s13048-021-00857-2 pmid: 34454554
[20]	Lv Y, Guo S, Li XG, et al. Sputum and serum microRNA-144 levels in patients with tuberculosis before and after treatment. Int J Infect Dis, 2016, 43(1):68-73. doi:10.1016/j.ijid.2015.12.014. doi: 10.1016/j.ijid.2015.12.014 URL
[21]	彭乐, 李萌, 席向宇. miR-144调控Atg4a在巨噬细胞清除结核分枝杆菌中的作用研究. 预防医学情报杂志, 2020, 36(12):1621-1628.
[22]	张立营, 陈朴, 高鹏, 等. 活动期肺结核外周血mir-144的表达水平及意义. 医学研究杂志, 2017, 46(1):151-154. doi:10.11969/j.issn.1673-548X.2017.01.040. doi: 10.11969/j.issn.1673-548X.2017.01.040

分组	LncRNA GAS5	miR-144-3p
NC组	1.00±0.00	1.00±0.00
MTB组	0.22±0.02^a	2.46±0.13^a
MTB+pcDNA组	0.23±0.01	2.48±0.14
MTB+pcDNA-GAS5组	0.86±0.07^bc	1.35±0.10^bc
MTB+inhibitor NC组	0.24±0.02	2.47±0.12
MTB+miR-144-3p inhibitor组	0.23±0.01	1.21±0.09^be
MTB+pcDNA-GAS5+mimic NC组	0.87±0.08	1.36±0.11
MTB+pcDNA-GAS5+miR-144-3p mimic组	0.88±0.07	1.89±0.14^df
F值	365.816	186.249
P值	<0.001	<0.001

分组	IFN-γ	IL-6	TNF-α
NC组	35.58±1.23	25.46±1.18	51.12±2.03
MTB组	212.26±9.65^a	123.35±5.12^a	325.58±12.28^a
MTB+pcDNA组	210.63±9.78	125.52±4.86	327.73±10.15
MTB+pcDNA-GAS5组	96.63±4.17^bc	41.93±2.19^bc	118.85±6.03^bc
MTB+inhibitor NC组	214.45±10.03	126.05±4.77	326.69±8.33
MTB+miR-144-3p inhibitor组	103.36±5.11^be	39.95±1.62^be	120.26±6.67^be
MTB+pcDNA-GAS5+mimic NC组	95.51±4.16	40.95±2.05	116.64±5.84
MTB+pcDNA-GAS5+miR-144-3p mimic组	152.26±6.78^df	85.53±4.11^df	249.92±10.05^df
F值	551.964	905.404	1167.700
P值	<0.001	<0.001	<0.001

分组	A₄₅₀值	细胞凋亡率(%)
NC组	0.86±0.07	9.79±0.23
MTB组	1.45±0.14^a	3.84±0.24^a
MTB+pcDNA组	1.44±0.13	3.82±0.21
MTB+pcDNA-GAS5组	0.96±0.08^bc	7.62±0.17^bc
MTB+inhibitor NC组	1.46±0.12	3.86±0.22
MTB+miR-144-3p inhibitor组	0.92±0.08^be	7.51±0.19^be
MTB+pcDNA-GAS5+mimic NC组	0.97±0.08	7.66±0.13
MTB+pcDNA-GAS5+miR-144-3p mimic组	1.25±0.13^df	5.46±0.16^df
F值	36.128	795.234
P值	<0.001	<0.001

分组	Bax/GAPDH	Bcl-2/GAPDH
NC组	1.21±0.12	0.56±0.05
MTB组	0.22±0.02^a	1.53±0.15^a
MTB+pcDNA组	0.23±0.01	1.54±0.14
MTB+pcDNA-GAS5组	0.94±0.08^bc	0.81±0.07^bc
MTB+inhibitor NC组	0.24±0.02	1.54±0.12
MTB+miR-144-3p inhibitor组	0.89±0.08^be	0.78±0.07^be
MTB+pcDNA-GAS5+mimic NC组	0.96±0.07	0.79±0.07
MTB+pcDNA-GAS5+miR-144-3p mimic组	0.58±0.05^df	1.08±0.11^df
F值	209.594	91.356
P值	<0.001	<0.001