TGFBR2 accelerates the oxidative stress and inflammation in septic acute lung injury via METTL14-mediated m6A modification or USP7-regulated deubiquitination

Background: Acute lung injury (ALI) caused by sepsis is a serious complication of sepsis and a major cause of death, and there is a lack of effective drug treatment. Transforming growth factor beta receptor II (TGFBR2) expression is related to sepsis and acute lung injury. Therefore, this study is to clarify the mechanism of TGFBR2 in sepsis-induced ALI. Methods: The study stimulated human pulmonary microvascular endothelial cells (HPMECs) using lipopolysaccharides (LPS) to establish an in vitro ALI model. The protein and mRNA levels were detected using western blot and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. The cell viability and proliferation were assessed using cell counting kit-8 (CCK8) and 5-ethynyl-2’-deoxyuridine (EdU) assays, respectively. Flow cytometry was conducted to analyze cell apoptosis. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the interleukin (IL)-6, IL-1β, and tumor necrosis factor-alpha (TNF-α) levels. The reactive oxygen species (ROS) and malondialdehyde (MDA) levels were examined using corresponding detection kits. Bioinformatic analysis was used to predict the N6-methyladenosine (m6A) methylation modifications and the interaction between TGFBR2 and methyltransferase 14 (METTL14)/ubiquitin-specific protease 7 (USP7). RNA immunoprecipitation (RIP), methylated RNA immunoprecipitation (MeRIP), and dual-luciferase reporter gene assay were used to identify the association of TGFBR2 with METTL14 and insulin like growth factor 2 mRNA binding protein 2 (IGF2BP2). Besides, the ubibrowser database, co-immunoprecipitation (Co-IP), and deubiquitination assays were performed on the relationship between USP7 and TGFBR2. Finally, a mouse model of polymicrobial sepsis was established to analyze the effects of TGFBR2 on lung injury in vivo . Results: TGFBR2 levels were highly expressed in the serum of sepsis-ALI patients and LPS-induced HPMECs. TGFBR2 knockdown remitted LPS-induced inhibition of viability and proliferation, as well as LPS-induced promoting effects on HPMEC apoptosis, inflammation, and oxidative stress. METTL14 and IGF2BP2 stabilized TGFBR2 mRNA expression through m6A methylation modification. Furthermore, silencing METTL14 protected HPMECs from LPS-induced injury by decreasing TGFBR2 expression. USP7 could stabilize the expression of TGFBR2 via deubiquitination, and si-USP7 ameliorated LPS-induced HPMEC damage via inhibiting TGFBR2 expression. TGFBR2 knockdown alleviated sepsis-induced ALI in vivo . Conclusion: TGFBR2 facilitates the inflammation and oxidative stress in sepsis-induced ALI via METTL14/IGF2BP2-mediated m6A modification or USP7-regulated deubiquitination. These findings provide a novel potential therapeutic target for the treatment of sepsis-induced ALI.