Protective effect of FOXP3-mediated miR-146b-5p/Robo1/NF-κB system on lipopolysaccharide-induced acute lung injury in mice.

Original Article Page 1 of 12 Protective effect of FOXP3-mediated miR-146b-5p/Robo1/NF-κB system on lipopolysaccharide-induced acute lung injury in mice Jiang Zhu1, Gaoli Chen2 1 Department of Respiratory and Critical Care Medicine, Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, University Hospital of Electronic Science & Technology of China, Chengdu, China; 2Department of Blood Transfusion, Teaching Hospital of Chengdu University of TCM, Chengdu, China Contributions: (I) Conception and design: All authors; (II) Administrative support: All authors; (III) Provision of study materials or patients: All authors; (IV) Collection and assembly of data: All authors; (V) Data analysis and interpretation: All authors; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. Correspondence to: Gaoli Chen. Department of Blood Transfusion, Teaching Hospital of Chengdu University of TCM, No.39, Shierqiao Road, Jinniu District, Chengdu 610072, China. Email: . Background: As a key transcription factor, forkhead box protein 3 (FOXP3) plays an important role in the development and function of natural cluster of differentiation 4 [CD4 (+)] regulatory T cells (Treg cells). However, the function of FOXP3 in Lipopolysaccharide (LPS)-induced acute lung injury (ALI) through regulating miR-146b-5p is unclear. This research aimed to disclose the regulatory effect of the FOXP3mediated miR-146b-5p/Roundabout 1 (Robo1)/NF-κB system on LPS-induced ALI in mice. Methods: The mice were subjected to 5 mg/kg of LPS via intratracheal instillation to induce ALI and generate the ALI model. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, the ALI + miR antagomir group and ALI + miR antagomir+ FOXP3 group. Lung tissue injury were detected by hematoxylin and eosin (HE) staining. Lung wet/dry weight ratio, total cells in bronchoalveolar lavage fluid (BALF), total protein in BALF and the polymorphonuclear leukocyte (PMN) in BALF were detected. The levels of tumor necrosis factor-α (TNF-α), Interleukin 6 (IL-6) and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA) kit. The dual-luciferase reporter assay were used to detect the target relationship between FOXP3 and Robo1. Mice was divided into five group, including control group, ALI group, ALI + FOXP3 group, ALI + Robo1 group and ALI + FOXP3+ Robo1 group. The protein levels of FOXP3, Robo1 and p-p65 were detected by western bolt. The mRNA levels of miR-146b5p and Robo1 were detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: Although protein expression levels of FOXP3 were significantly down-regulated in the ALI model, the increased FOXP3 levels promoted an increase in miR-146b-5p. Compared with the control group, the ALI model group exhibited severe histopathologic injury, such as thickening of the alveolar wall, pulmonary congestion, and decreased alveolar numbers. By mediating the overexpression of miR146b-5p, FOXP3 also increased alveolar clearance and inhibited inflammatory responses in the ALI model. Importantly, Robo1 is a potential target of miR-146b-5p. Conclusions: FOXP3 could inhibit NF-κB activation, reduce lung pathological damage, and inhibit inflammatory responses by mediating the miR-146b-5p/Robo1/NF-κB system in the ALI model. These results may provide a new potential target for the treatment of ALI disease. Keywords: FOXP3; LPS-induced acute lung injury; miR-146b-5p/Robo1/NF-κB Submitted Oct 16, 2020. Accepted for publication Dec 13, 2020. doi: 10.21037/atm-20-7703 View this article at: http://dx.doi.org/10.21037/atm-20-7703 © Annals of Translational Medicine. All rights reserved. Ann Transl Med 2020;8(24):1651 | http://dx.doi.org/10.21037/atm-20-7703 Page 2 of 12 Introduction Acute respiratory distress syndrome (ARDS) is a clinically important complication of severe acute lung injury (ALI). Infections such as sepsis and pneumonia are the leading causes of ALI/ARDS (1,2), and histology shows the pulmonary manifestations of an acute systemic inflammatory process characterized by pulmonary infiltrates, hypoxaemia, and oedema. As many as 25 cases per 100,000 are reported annually, with a high prevalence in young people (3,4). The Lipopolysaccharide (LPS)-induced ALI model is often used to examine lung injury and many components of its response, particularly the acute pathways. At the same time, LPS is an effective activator of the innate immune pathway, greatly imitating the pathological changes of ALI (5,6). Regulatory T (Treg) cell subsets have specific transcription fork head box protein 3 (FOXP3), a unique cell type that maintains immune homeostasis by controlling the response of effector T (Teff) cells. Research has shown that FOXP3 is also called fork head/winged helix transcription factor (FOXP3). FOXP3 is specifically expressed on cluster of differentiation (CD)4+CD25+ regulatory T cells and is a key transcription factor for its production and development (7). The regulation and proliferation rate of lung epithelial cells following lung injury is strongly correlated with the number of FOXP3 + Treg CD103 (8). One report indicates that FOXP3 expression in Tregs may be down-regulated in the inflammatory alveolar microenvironment by LPS-induced ALI (9). This raises the question of whether FOXP3 plays a protective role in LPS-induced ALI models. Diverse biological processes have been associated with miRNAs, such as inducing M2 polarization (10), regulating insulin secretion (11), and suppressing cancer invasion (12,13). Further, miR146 has been associated with the control of inflammatory responses (14), inducing the differentiation of macrophages into M2 cells (15), inhibiting cancer cell metastasis (16), and brake myeloproliferation (17). It is increasingly recognized that the human genome contains two miR-146 genes on chromosomes 5 and 10, respectively, including miR-146a (18) and miR-146b (19). Inflammation caused LPS-induced injury in A549 and H1975 cells and miR146 has been shown to relieve apoptosis (20). Importantly, FOXP3-induced mir-146 a/b has been displayed to not only restrain breast cancer cell proliferation and promote apoptosis, but also reversely regulate activation of NF-κB by inhibiting the expression of tumors necrosis factor receptor related factor 6 (TRAF6) and receptorrelated kinases (IRAK1) (21). Previous research has exhibited that miR-146b-5p © Annals of Translational Medicine. All rights reserved. Zhu and Chen. FOXP3 play a function in acute lung injury protects oligodendrocyte precursor cells from oxygen/ glucose deprivation-induced injury through regulating Keap1/Nrf2 signaling via targeting bromodomaincontaining protein 4 (22). MiR-146b-5p attenuates the inflammatory response of glomerular mesangial cells by inhibiting the expressions levels of TRAF6 and IRAK1 in lupus nephritis (23). p16INK4a inhibits the proliferation of osteosarcoma cells through r (...truncated)