MiR-203 targets TLR4 to regulate NF-κB/NLRP3 pathway to protect alveolar epithelial cells from LPS-induced injury_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

TANG Jianhua ¹ , LIU Jianhua ² , XU Tao ³ , LIU Keqin ¹ , ZHANG Heming ¹ ,  ZHANG Zhihua ²

1. Department of Pharmacy, The First Affiliated Hospital of Hebei North University, Zhangjiakou, Hebei 075000, P. R. China;
2. Department of Respiratory Medicine, The First Affiliated Hospital of Hebei North University, Zhangjiakou, Hebei 075000, P. R. China;
3. Department of Cardiology, The First Affiliated Hospital of Hebei North University, Zhangjiakou, Hebei 075000, P. R. China;

Corresponding author：

ZHANG Zhihua, Email: zzh19641229@163.com

Keywords：

microRNA-203; alveolar epithelial cells; lipopolysaccharide; cell apoptosis; inflammatory response

DOI：

10.7507/1671-6205.202110041

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Objective To explore whether microRNA-203 (miR-203) targets and regulates the Toll-like receptor 4 (TLR4)/nuclear transcription factor kappa B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) to protect alveolar epithelial cells from lipopolysaccharide (LPS)-induced apoptosis and inflammation injury. Methods The alveolar epithelial A549 cells were used as the research objects and divided into: Control group (normal culture), LPS group (LPS treatment), LPS+miR-NC mimics group (LPS treatment after transfection of miR-NC mimics), LPS+ miR-203 mimics group (LPS treatment after transfection of miR-203 mimics), LPS+miR-203 mimics+pcDNA group (LPS treatment after transfection of miR-203 mimics and pcDNA), LPS+miR-203 mimics+pcDNA-TLR4 group (LPS treatment after transfection of miR-203 mimics and pcDNA-TLR4). Dual luciferase reporter gene was used to detect the targeting relationship between miR-203 and TLR4; Real-time quantitative reverse transcription-polymerase chain reaction was used to detect the relative expression levels of miR-203 and TLR4 mRNA; enzyme-linked immunosorbent assay was used to measure the levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6; flow cytometry was used to detect the apoptosis rate of A549 cells; Western blot was used to detect the expression of B-cell lymphoma/leukemia-2 gene (Bcl-2) and Bcl-2 associated X protein (Bax), TLR4, NF-κB and NLRP3 proteins in A549 cells. Results There was a targeted regulation relationship between miR-203 and TLR4. Compared with the Control group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the level of Bcl-2 protein in cells decreased (P<0.05). Compared with the LPS+miR-NC mimics group, the expression of TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics group decreased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant decreased, the apoptosis rate decreased, the expression level of miR-203 and the level of Bcl-2 protein in cells increased (P<0.05). Compared with the LPS+miR-203 mimics+pcDNA group, the expression of miR-203, TLR4 mRNA and protein, Bax, NF-κB, and NLRP3 proteins in A549 cells in the LPS+miR-203 mimics+pcDNA-TLR4 group increased, the levels of TNF-α, IL-1β and IL-6 in the cell supernatant increased, the apoptosis rate increased, the expression level of miR-203 and the level of Bcl-2 protein in cells decreased (P<0.05). Conclusion MiR-203 can target TLR4/NF-κB/NLRP3 to protect alveolar epithelial cells from apoptosis and inflammation induced by LPS.

Citation： TANG Jianhua, LIU Jianhua, XU Tao, LIU Keqin, ZHANG Heming, ZHANG Zhihua. MiR-203 targets TLR4 to regulate NF-κB/NLRP3 pathway to protect alveolar epithelial cells from LPS-induced injury. Chinese Journal of Respiratory and Critical Care Medicine, 2022, 21(6): 425-431. doi: 10.7507/1671-6205.202110041 Copy

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2.	Abedi F, Hayes AW, Reiter R, et al. Acute lung injury: the therapeutic role of Rho kinase mimicss. Pharmacol Res, 2020, 155(2): 104736-104742.
3.	Chandan K, Gupta M, Sarwat M. Role of host and pathogen-derived microRNAs in immune regulation during infectious and inflammatory diseases. Front Immunol, 2020, 10(2): 3081-3084.
4.	Liu HP, Zhang Y, Liu ZT, et al. MiR-203 regulates proliferation and apoptosis of ovarian cancer cells by targeting SOCS3. Eur Rev Med Pharmacol Sci, 2020, 24(15): 7914-7922.
5.	李建伟, 张丽娜, 张英民, 等. miR-203基因启动子区甲基化与肥胖膝关节骨性关节炎患者滑膜炎症水平相关性研究. 中国医药导报, 2020, 542(12): 38-41+66.
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8.	Ke XF, Fang J, Wu XN, et al. MicroRNA-203 accelerates apoptosis in LPS-stimulated alveolar epithelial cells by targeting PIK3CA. Biochem Biophys Res Commun, 2014, 450(4): 1297-1303.
9.	亓水芹, 冯向功. miR-221靶向AdipoR1基因对LPS诱导的肺泡上皮细胞A549炎症分泌及凋亡影响. 中国免疫学杂志, 2020, 36(5): 538-542,554.
10.	Liu J, Dean DA. Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function. Sci Rep, 2021, 11(1): 20862-20877.
11.	Han J, Li H, Bhandari S, et al. Maresin conjugates in tissue regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury. J Cell Mol Med, 2020, 24(8): 4736-4747.
12.	Azouz F, Arora K, Krause K, et al. Integrated microRNA and mRNA profiling in Zika Virus-Infected Neurons. Viruses, 2019, 11(2): 162-183.
13.	Chu Q, Han JJ, Sun LP, et al. Characterization of MDA5 and microRNA-203 negatively regulates the RLR signaling pathway via targeting MDA5 in miiuy croaker. Dev Comp Immunol, 2022, 126(1): 104259.
14.	Li Y, Liu XP, Du AL, et al. miR-203 accelerates apoptosis and inflammation induced by LPS via targeting NFIL3 in cardiomyocytes. J Cell Biochem, 2019, 120(4): 6605-6613.
15.	Luo X, O'Neill KL, Huang K. The third model of Bax/Bak activation: a Bcl-2 family feud finally resolved?. F1000Res, 2020, 9(Faculty Rev).
16.	任依梦, 房静远. Toll样受体家族相关信号通路及其与结直肠癌关系的研究进展. 肿瘤, 2019, 39(7): 589-594.
17.	秦臻, 汪勃, 谭赵霞, 等. 姜黄素抑制 TLR4/HMGB1 通路保护脂多糖诱导急性肺损伤的作用. 中国胸心血管外科临床杂志, 2020, 27(6): 685-688.
18.	Somensi N, Rabelo TK, Guimarães AG, et al. Carvacrol suppresses LPS-induced pro-inflammatory activation in RAW 264.7 macrophages through ERK1/2 and NF-κB pathway. Int Immunopharmacol, 2019, 75(2): 105743-105752.
19.	古丽菲热·塔依尔, 杨春波, 李祥, 等. 脓毒症肠道损伤模型中NLRP3炎症小体活化介导炎症反应及细胞凋亡. 中华危重病急救医学, 2021, 33(7): 855-860.
20.	Yan YR, Lu KX, Ye T, et al. MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB. Int J Mol Med, 2019, 43(3): 1467-1477.
21.	Gan WH, Li XH, Cui YY, et al. Pinocembrin relieves lipopolysaccharide and bleomycin induced lung inflammation via inhibiting TLR4-NF-κB-NLRP3 inflammasome signaling pathway. Int Immunopharmacol, 2021, 90(3): 107230.

1. Wu XJ, Kong Q, Zhan LY, et al. TIPE2 ameliorates lipopolysaccharide-induced apoptosis and inflammation in acute lung injury. Inflamm Res, 2019, 68(11): 981-992.
2. Abedi F, Hayes AW, Reiter R, et al. Acute lung injury: the therapeutic role of Rho kinase mimicss. Pharmacol Res, 2020, 155(2): 104736-104742.
3. Chandan K, Gupta M, Sarwat M. Role of host and pathogen-derived microRNAs in immune regulation during infectious and inflammatory diseases. Front Immunol, 2020, 10(2): 3081-3084.
4. Liu HP, Zhang Y, Liu ZT, et al. MiR-203 regulates proliferation and apoptosis of ovarian cancer cells by targeting SOCS3. Eur Rev Med Pharmacol Sci, 2020, 24(15): 7914-7922.
5. 李建伟, 张丽娜, 张英民, 等. miR-203基因启动子区甲基化与肥胖膝关节骨性关节炎患者滑膜炎症水平相关性研究. 中国医药导报, 2020, 542(12): 38-41+66.
6. Li C, Ma DX, Zhou HC, et al. Effects of different doses lipopolysaccharides on the mucosal barrier in mouse intestine. Res Vet Sci, 2020, 133(2): 75-84.
7. 王凯, 刘小虹. 喘可治对小鼠急性肺损伤TLR4/NF-κB/NLRP3通路的影响. 中国实验方剂学杂志, 2017, 32(22): 151-156.
8. Ke XF, Fang J, Wu XN, et al. MicroRNA-203 accelerates apoptosis in LPS-stimulated alveolar epithelial cells by targeting PIK3CA. Biochem Biophys Res Commun, 2014, 450(4): 1297-1303.
9. 亓水芹, 冯向功. miR-221靶向AdipoR1基因对LPS诱导的肺泡上皮细胞A549炎症分泌及凋亡影响. 中国免疫学杂志, 2020, 36(5): 538-542,554.
10. Liu J, Dean DA. Gene transfer of MRCKα rescues lipopolysaccharide-induced acute lung injury by restoring alveolar capillary barrier function. Sci Rep, 2021, 11(1): 20862-20877.
11. Han J, Li H, Bhandari S, et al. Maresin conjugates in tissue regeneration 1 improves alveolar fluid clearance by up-regulating alveolar ENaC, Na, K-ATPase in lipopolysaccharide-induced acute lung injury. J Cell Mol Med, 2020, 24(8): 4736-4747.
12. Azouz F, Arora K, Krause K, et al. Integrated microRNA and mRNA profiling in Zika Virus-Infected Neurons. Viruses, 2019, 11(2): 162-183.
13. Chu Q, Han JJ, Sun LP, et al. Characterization of MDA5 and microRNA-203 negatively regulates the RLR signaling pathway via targeting MDA5 in miiuy croaker. Dev Comp Immunol, 2022, 126(1): 104259.
14. Li Y, Liu XP, Du AL, et al. miR-203 accelerates apoptosis and inflammation induced by LPS via targeting NFIL3 in cardiomyocytes. J Cell Biochem, 2019, 120(4): 6605-6613.
15. Luo X, O'Neill KL, Huang K. The third model of Bax/Bak activation: a Bcl-2 family feud finally resolved?. F1000Res, 2020, 9(Faculty Rev).
16. 任依梦, 房静远. Toll样受体家族相关信号通路及其与结直肠癌关系的研究进展. 肿瘤, 2019, 39(7): 589-594.
17. 秦臻, 汪勃, 谭赵霞, 等. 姜黄素抑制 TLR4/HMGB1 通路保护脂多糖诱导急性肺损伤的作用. 中国胸心血管外科临床杂志, 2020, 27(6): 685-688.
18. Somensi N, Rabelo TK, Guimarães AG, et al. Carvacrol suppresses LPS-induced pro-inflammatory activation in RAW 264.7 macrophages through ERK1/2 and NF-κB pathway. Int Immunopharmacol, 2019, 75(2): 105743-105752.
19. 古丽菲热·塔依尔, 杨春波, 李祥, 等. 脓毒症肠道损伤模型中NLRP3炎症小体活化介导炎症反应及细胞凋亡. 中华危重病急救医学, 2021, 33(7): 855-860.
20. Yan YR, Lu KX, Ye T, et al. MicroRNA-223 attenuates LPS-induced inflammation in an acute lung injury model via the NLRP3 inflammasome and TLR4/NF-κB signaling pathway via RHOB. Int J Mol Med, 2019, 43(3): 1467-1477.
21. Gan WH, Li XH, Cui YY, et al. Pinocembrin relieves lipopolysaccharide and bleomycin induced lung inflammation via inhibiting TLR4-NF-κB-NLRP3 inflammasome signaling pathway. Int Immunopharmacol, 2021, 90(3): 107230.

Chinese Journal of Respiratory and Critical Care Medicine

MiR-203 targets TLR4 to regulate NF-κB/NLRP3 pathway to protect alveolar epithelial cells from LPS-induced injury

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