LPS-induced TMBIM6 splicing drives endothelial necroptosis and aggravates ALI.

Abstract

Background: The mechanism underlying necroptosis in pulmonary vessel endothelial cells (PVECs) resulting from long non-coding RNA (lncRNA)-induced alternative splicing (AS) of target genes in acute lung injury (ALI) remains unclear.

Methods: Lipopolysaccharide (LPS)-induced expression of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and lncRNAs was analyzed via RT-PCR in PVECs. Full-transcriptome sequencing was used to detect AS-related mRNAs. The interaction between lncRNA MALAT1 and target gene transmembrane BAX inhibitor motif-containing 6 (TMBIM6) was verified using a dual-luciferase reporter system. Necroptosis was measured as protein levels of phosphorylated receptor-interacting serine/threonine kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like (MLKL) proteins, as well as flow cytometer measurement. Antisense of MALAT1, TMBIM6, TMBIM6-225 and RIPK1 inhibitor were transfected into a rat model of LPS-induced ALI. Hematoxylin and eosin (H&E) and immunohistochemical staining were performed to evaluate lung injury.

Results: LPS upregulated the expression of TNF-α, IL-1β, IL-6, p-RIPK1, p-RIPK3, p-MLKL, MALAT1, and TMBIM6-225 (an AS isoform of MALAT1-targeted gene TMBIM6) in PVECs. However, it downregulated the expression of TMBIM6. An antisense of MALAT1 inhibited TMBIM6-225 and downregulated p-MLKL. The pro-necroptotic effect of MALAT1 was verified in an LPS-induced MALAT1/shMALAT1-transfected ALI rat model in vivo. The necroptotic effect was reversed by treatment with necrostatin-1.

Conclusions: LPS-induced MALAT1 causes AS of TMBIM6, and the AS variant TMBIM6-225 aggravates ALI by promoting PVEC necroptosis via the p-RIPK1, p-RIPK3, and p-MLKL complex.