N6-methyladenosine modification of CBLB affects LPS-induced endoplasmic reticulum stress in alveolar epithelial cells

Introduction
Acute lung injury (ALI) is defined as an acute hypoxic respiratory insufficiency that arises from injury to alveolar epithelial cells. The suppression of endoplasmic reticulum stress (ERS) was reported to protect against ALI. This study investigated the mechanism of N6-methyladenosine (m6A)-modified Casitas B lymphoma-b (CBLB) on ERS in lipopolysaccharide (LPS)-induced type II alveolar epithelial cells (AECIIs).

Material and methods
AECII viability was evaluated via Cell Counting Kit-8 (CCK-8). The reactive oxygen species (ROS) level was examined via an ROS kit. TUNEL staining was applied to assay apoptosis. The levels of CBLB, LIM domain kinase 1 (LIMK1), methyltransferase-like 14 (METTL14), and ERS-related proteins were assessed by western blot and immunofluorescence. The ubiquitination level of LIMK1 was analyzed using immunoprecipitation. The interrelationship between CBLB and LIMK1 was identified via Co-IP. The m6A modification of CBLB was analyzed through MeRIP. The binding of METTL14 to the CBLB mRNA was verified by RIP.

Results
CBLB facilitated the ubiquitination and degradation of LIMK1. The suppressive action of upregulated CBLB on apoptosis and ERS in LPS-induced AECIIs was reversed by LIMK1 overexpression. METTL14 reduced the stability of CBLB mRNA in a manner dependent on m6A methylation. CBLB knockdown reversed the inhibitory effects of METTL14 silencing on apoptosis and ERS in LPS-stimulated AECIIs.

Conclusions
METTL14-mediated m6A modification of CBLB impaired the mRNA stability of CBLB to block the degradation of LIMK1, which promoted ERS in LPS-induced AECIIs.