Effects of LncRNA-NORAD on acute lung injury in septic rats by regulating the miR-155-5p/TLR6 molecular axis_Chinese Journal of Respiratory and Critical Care Medicine

Authors：

 ZENG Haiwen ¹ , CHEN Qiaoli ² ,  DING Zhirong ¹

1. Department of Critical Care Medicine, Fujian Medical University Affiliated Quanzhou First Hospital, Quanzhou, Fujian 362000, P. R.China;
2. Comprehensive Ward, Fujian Medical University Affiliated Quanzhou First Hospital, Quanzhou, Fujian 362000, P. R.China;

Corresponding author：

DING Zhirong, Email: 13385951978@189.com

Keywords：

Sepsis; acute lung injury; non-coding RNA activated by DNA damage; miR-155-5p/TLR6

DOI：

10.7507/1671-6205.202402017

Video：

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Objective To investigate the effect of non-coding RNA activated by DNA damage (NORAD) on acute lung injury (ALI) in septic rats by regulating the miR-155-5p/TLR6 molecular axis. Methods The rats were randomly divided into control group, model group, low NORAD expression no-load group (LV-sh-NC), low NORAD expression group (LV-sh-NORAD), low NORAD expression +miR-155-5p low expression no-load group (LV-sh-NORAD+NC antagomir), NORAD low expression +miR-155-5p low expression group (LV-sh-NORAD+miR-155-5p antagomir). ELISA kits were applied to detect interleukin (IL)-8, IL-1β, and tumor necrosis factor-α (TNF-α) levels; quantitative real-time polymerase chain reaction was applied to detect the expression of NORAD, miR-155-5p, and Toll-like receptor 6 (TLR6) genes in lung tissue of rats in each group. The ratio of wet weight to dry weight (W/D) of lung tissue was measured. The pathological changes of lung tissue were observed by hematoxylin-eosin staining, and apoptosis in lung tissue cells was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling. Western blot was applied to detect the expressions of TLR6, Bax, Bcl-2, and cleaved cysteinyl aspartate specific proteinase 3 caspase-3) proteins in cells. Dual luciferase reporter gene experiment was applied to verify the relationship between miR-155-5p and NORAD and TLR6. Results Compared with the control group, the lung tissue of rats in the model group and LV-sh-NC group was obviously damaged, the levels of serum IL-1β, TNF-α, IL-8, expression of NORAD and TLR6 mRNA in lung tissue, W/D ratio, apoptosis rate, expression of TLR6, Bax, and Cleaved-caspase-3 proteins were obviously increased, the expression of miR-155-5p and Bcl-2 proteins in lung tissue was obviously reduced (P<0.05). Down-regulation of NORAD expression could reduce lung tissue injury, serum IL-1β, TNF-α, IL-8 levels, mRNA expression of NORAD and TLR6 in lung tissue, W/D ratio, apoptosis rate, TLR6, Bax, Cleaved caspase-3 protein expression, and cleaved caspase-3 protein expression. The expression of miR-155-5p and Bcl-2 protein in lung tissue were significantly increased (P<0.05). Down-regulating the expression of miR-155-5p could reduce the improvement effect of negatively regulated NORAD on sepsis ALI rats (P<0.05). Conclusion Interference with NORAD can alleviate lung injury in ALI rats by regulating the miR-155-5p/TLR6 molecular axis.

Citation： ZENG Haiwen, CHEN Qiaoli, DING Zhirong. Effects of LncRNA-NORAD on acute lung injury in septic rats by regulating the miR-155-5p/TLR6 molecular axis. Chinese Journal of Respiratory and Critical Care Medicine, 2025, 24(1): 26-32. doi: 10.7507/1671-6205.202402017 Copy

1.	Zhang H, Liu J, Zhou Y, et al. Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci, 2022, 18(8): 3337-3357.
2.	Gong F, Li R, Zheng X, et al. OLFM4 regulates lung epithelial cell function in sepsis-associated ARDS/ALI via LDHA-mediated NF-κB signaling. J Inflamm Res, 2021, 14(2): 7035-7051.
3.	Gao M, Yu T, Liu D, et al. Sepsis plasma-derived exosomal miR-1-3p induces endothelial cell dysfunction by targeting SERP1. Clin Sci (Lond), 2021, 135(2): 347-365.
4.	Zhang X, Cui Y, Ding X, et al. Analysis of mRNA-lncRNA and mRNA-lncRNA-pathway co-expression networks based on WGCNA in developing pediatric sepsis. Bioengineered, 2021, 12(1): 1457-1470.
5.	Zhou Y, Chen C, Li Q, et al. NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage. Open Med (Wars), 2022, 17(1): 676-688.
6.	Lei N, Kong P, Chen S, et al. Upregulated NORAD is implicated in apoptosis, inflammation, and oxidative stress in ulcerative colitis through the nuclear factor-κappaB signaling. Eur J Gastroenterol Hepatol, 2022, 34(6): 630-639.
7.	Hao X, Wei H. LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis. BMC Pulm Med, 2022, 22(1): 371-382.
8.	Lin X, Yu T, Luo J, et al. BMSCs mediates endothelial cell autophagy by upregulating miR-155-5p to alleviate ventilator-induced lung injury. J Biochem Mol Toxicol, 2022, 36(7): e23060-e23072.
9.	Wang HF, Li Y, Wang YQ, et al. MicroRNA-494-3p alleviates inflammatory response in sepsis by targeting TLR6. Eur Rev Med Pharmacol Sci, 2019, 23(7): 2971-2977.
10.	谢小芳, 赵展庆, 符妹垂. 丁香苷调节NLRP3/Caspase-1信号通路对脓毒症肺损伤大鼠细胞焦亡的影响. 中国药学杂志, 2023, 58(19): 1744-1751.
11.	Qu M, Chen Z, Qiu Z, et al. Neutrophil extracellular traps-triggered impaired autophagic flux via METTL3 underlies sepsis-associated acute lung injury. Cell Death Discov, 2022, 8(1): 375-384.
12.	Cao Z, Qin H, Huang Y, et al. Crosstalk of pyroptosis, ferroptosis, and mitochondrial aldehyde dehydrogenase 2-related mechanisms in sepsis-induced lung injury in a mouse model. Bioengineered, 2022, 13(3): 4810-4820.
13.	Li J, Xu H, Li N, et al. Long non-coding RNA growth arrest specific 5 is downregulated in sepsis-ALI and inhibits apoptosis by up-regulating miR-146a. Bioengineered, 2022, 13(2): 4146-4152.
14.	Miao RF, Tu J. LncRNA CDKN2B-AS1 interacts with LIN28B to exacerbate sepsis-induced acute lung injury by inducing HIF-1α/NLRP3-mediated pyroptosis. Kaohsiung J Med Sci, 2023, 39(9): 883-895.
15.	Xie Z, Wei L, Chen J, et al. LncRNA NORAD deficiency alleviates kidney injury in mice and decreases the inflammatory response and apoptosis of lipopolysaccharide-stimulated HK-2 cells via the miR-577/GOLPH3 axis. Cytokine, 2022, 153(2): 155844-155856.
16.	Zhang H, Li L, Xu L, et al. Clinical significance of the serum lncRNA NORAD expression in patients with neonatal sepsis and its association with miR-410-3p. J Inflamm Res, 2021, 14(3): 4181-4188.
17.	Liu X, Gao C, Wang Y, et al. BMSC-Derived Exosomes Ameliorate LPS-induced acute lung injury by miR-384-5p-controlled alveolar macrophage autophagy. Oxid Med Cell Longev, 2021, 2021(2): 9973457-9973468.
18.	Yi X, Wei X, Lv H, et al. Exosomes derived from microRNA-30b-3p-overexpressing mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting SAA3. Exp Cell Res, 2020, 394(1): 112160-112171.
19.	Kataoka H, Saeki A, Hasebe A, et al. Naringenin suppresses Toll-like receptor 2-mediated inflammatory responses through inhibition of receptor clustering on lipid rafts. Food Sci Nutr, 2020, 9(2): 963-972.
20.	Zou M, Yang L, Niu L, et al. Baicalin ameliorates mycoplasma gallisepticum-induced lung inflammation in chicken by inhibiting TLR6-mediated NF-κB signalling. Br Poult Sci, 2021, 62(2): 199-210.
21.	Khan MJ, Singh P, Jha P, et al. Investigating the link between miR-34a-5p and TLR6 signaling in sepsis-induced ARDS. 3 Biotech, 2023, 13(8): 282-293.

1. Zhang H, Liu J, Zhou Y, et al. Neutrophil extracellular traps mediate m6A modification and regulates sepsis-associated acute lung injury by activating ferroptosis in alveolar epithelial cells. Int J Biol Sci, 2022, 18(8): 3337-3357.
2. Gong F, Li R, Zheng X, et al. OLFM4 regulates lung epithelial cell function in sepsis-associated ARDS/ALI via LDHA-mediated NF-κB signaling. J Inflamm Res, 2021, 14(2): 7035-7051.
3. Gao M, Yu T, Liu D, et al. Sepsis plasma-derived exosomal miR-1-3p induces endothelial cell dysfunction by targeting SERP1. Clin Sci (Lond), 2021, 135(2): 347-365.
4. Zhang X, Cui Y, Ding X, et al. Analysis of mRNA-lncRNA and mRNA-lncRNA-pathway co-expression networks based on WGCNA in developing pediatric sepsis. Bioengineered, 2021, 12(1): 1457-1470.
5. Zhou Y, Chen C, Li Q, et al. NORAD modulates miR-30c-5p-LDHA to protect lung endothelial cells damage. Open Med (Wars), 2022, 17(1): 676-688.
6. Lei N, Kong P, Chen S, et al. Upregulated NORAD is implicated in apoptosis, inflammation, and oxidative stress in ulcerative colitis through the nuclear factor-κappaB signaling. Eur J Gastroenterol Hepatol, 2022, 34(6): 630-639.
7. Hao X, Wei H. LncRNA H19 alleviates sepsis-induced acute lung injury by regulating the miR-107/TGFBR3 axis. BMC Pulm Med, 2022, 22(1): 371-382.
8. Lin X, Yu T, Luo J, et al. BMSCs mediates endothelial cell autophagy by upregulating miR-155-5p to alleviate ventilator-induced lung injury. J Biochem Mol Toxicol, 2022, 36(7): e23060-e23072.
9. Wang HF, Li Y, Wang YQ, et al. MicroRNA-494-3p alleviates inflammatory response in sepsis by targeting TLR6. Eur Rev Med Pharmacol Sci, 2019, 23(7): 2971-2977.
10. 谢小芳, 赵展庆, 符妹垂. 丁香苷调节NLRP3/Caspase-1信号通路对脓毒症肺损伤大鼠细胞焦亡的影响. 中国药学杂志, 2023, 58(19): 1744-1751.
11. Qu M, Chen Z, Qiu Z, et al. Neutrophil extracellular traps-triggered impaired autophagic flux via METTL3 underlies sepsis-associated acute lung injury. Cell Death Discov, 2022, 8(1): 375-384.
12. Cao Z, Qin H, Huang Y, et al. Crosstalk of pyroptosis, ferroptosis, and mitochondrial aldehyde dehydrogenase 2-related mechanisms in sepsis-induced lung injury in a mouse model. Bioengineered, 2022, 13(3): 4810-4820.
13. Li J, Xu H, Li N, et al. Long non-coding RNA growth arrest specific 5 is downregulated in sepsis-ALI and inhibits apoptosis by up-regulating miR-146a. Bioengineered, 2022, 13(2): 4146-4152.
14. Miao RF, Tu J. LncRNA CDKN2B-AS1 interacts with LIN28B to exacerbate sepsis-induced acute lung injury by inducing HIF-1α/NLRP3-mediated pyroptosis. Kaohsiung J Med Sci, 2023, 39(9): 883-895.
15. Xie Z, Wei L, Chen J, et al. LncRNA NORAD deficiency alleviates kidney injury in mice and decreases the inflammatory response and apoptosis of lipopolysaccharide-stimulated HK-2 cells via the miR-577/GOLPH3 axis. Cytokine, 2022, 153(2): 155844-155856.
16. Zhang H, Li L, Xu L, et al. Clinical significance of the serum lncRNA NORAD expression in patients with neonatal sepsis and its association with miR-410-3p. J Inflamm Res, 2021, 14(3): 4181-4188.
17. Liu X, Gao C, Wang Y, et al. BMSC-Derived Exosomes Ameliorate LPS-induced acute lung injury by miR-384-5p-controlled alveolar macrophage autophagy. Oxid Med Cell Longev, 2021, 2021(2): 9973457-9973468.
18. Yi X, Wei X, Lv H, et al. Exosomes derived from microRNA-30b-3p-overexpressing mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting SAA3. Exp Cell Res, 2020, 394(1): 112160-112171.
19. Kataoka H, Saeki A, Hasebe A, et al. Naringenin suppresses Toll-like receptor 2-mediated inflammatory responses through inhibition of receptor clustering on lipid rafts. Food Sci Nutr, 2020, 9(2): 963-972.
20. Zou M, Yang L, Niu L, et al. Baicalin ameliorates mycoplasma gallisepticum-induced lung inflammation in chicken by inhibiting TLR6-mediated NF-κB signalling. Br Poult Sci, 2021, 62(2): 199-210.
21. Khan MJ, Singh P, Jha P, et al. Investigating the link between miR-34a-5p and TLR6 signaling in sepsis-induced ARDS. 3 Biotech, 2023, 13(8): 282-293.

Chinese Journal of Respiratory and Critical Care Medicine

Effects of LncRNA-NORAD on acute lung injury in septic rats by regulating the miR-155-5p/TLR6 molecular axis

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