Long noncoding RNA SNHG4 remits lipopolysaccharide-engendered inflammatory lung damage by inhibiting METTL3 - Mediated m6A level of STAT2 mRNA

Emerging evidence suggests that long non coding RNA (lncRNA) small nucleolar RNA host gene 4 (SNHG4) has become a new insight into lipopolysaccharide (LPS)-induced microglia inflammation, its role in neonatal pneumonia (NP) remains to be largely unrevealed.

This study reveals that SNHG4 promotes LPS induced inflammation in human lung fibroblasts and mouse lung tissues in vitro and in vivo by inhibiting METTL3-mediated m6A level of STAT2 mRNA, which may provide a potential therapeutic mechanism for NP.

RT-qPCR was used to determine the expression of SNHG4 and METTL3 in the serum from NP patients and normal volunteers, as well as in LPS treated-WI-38 cells. The SNHG4 overexpression vector (pcDNA-SNHG4) was transfected into LPS-treated cells. CCK-8, Transwell, annexin V-FITC/PI, ELISA and Western blot assays were used to determine cell proliferation, migration, apoptosis, contents of IL-6, TNF-α, SOD and MDA, as well as the expression levels of NF-κB pathway proteins, respectively. The enrichment of SNHG4 in the METTL3 promoter region was assessed with RIP assay. m6A quantitative analysis illustrated the m6A level with or without SNHG4 overexpression or METTL3 silencing. Bioinformatics analysis and RIP-PCR were used to predict and validate YTHDF1-mediated m6A levels on signal transducer and activator of transcription 2 (STAT2) mRNA in METTL3 inhibited cells. Then rescue experiments were performed to explore effects of SNHG4 and METTL3 or STAT2 on LPS-treated cell functions. Subsequently, in vivo functional experiments were performed to investigate the role of SNHG4 in LPS induced pneumonia in mice.

SNHG4 was downregulated, and METTL3 was upregulated in NP patients and LPS-treated cells. SNHG4 overexpression facilitated cell proliferation, migration and SOD concentration, as well as inhibited cell apoptosis and production of IL-6, TNF-α and MDA, and suppressed the expression of NF-κB pathway proteins. Mechanistically, SNHG4 bound with METTL3 and downregulated METTL3 expression. Besides, total m6A level was lower in the SNHG4 overexpressed or METTL3 inhibited cells. METTL3 interference reduced m6A levels of STAT2 mRNA, decreased STAT2 mRNA stability and promoted STAT2 translation level. Upregulation of METTL3 or STAT2 reversed the effects of SNHG4 overexpression on LPS-treated cell functions. Conclusions: This study reveals that SNHG4 promotes LPS induced inflammation in human lung fibroblasts and mouse lung tissues in vitro and in vivo by inhibiting METTL3-mediated m6A level of STAT2 mRNA, which may provide a potential therapeutic mechanism for NP.