The Role And Mechanism Of LncRNA Rsf1os2-encoded Micropeptides Regulating Macrophage Polarization In Allergic Asthma

Objective: This study aims to elucidate the mechanisms involved in the role of micropeptide encoded by lncRNA Rsf1os2 in allergic asthma through the regulation of macrophage polarization.Methods: In vitro isolation and culture of mouse bone marrow-derived macrophages BMDM.identification of small open reading frame s ORF and prediction of coding potential of differentially expressed lnc RNAs in mouse bone marrow-derived macrophages BMDM using bioinformatics;verification of GFP fluorescence expression of candidate lnc RNAs by constructing ORFGFPmut fusion plasmids to determine coding ability;verification of GFP fluorescence expression of candidate lnc RNAs by constructing ORF-Flag tag fusion plasmids of lncRNA Rsf1os2 and transfecting cells for expression validation of lncRNA Rsf1os2 encoding short peptide.The molecular characteristics of lncRNA Rsf1os2 were analyzed by UCSC database,and the short peptide encoded by lncRNA Rsf1os2 was synthesized in vitro and immunized in rabbits to obtain specific polyclonal antibodies.The expression of lncRNA Rsf1os2 with its encoded short peptide was verified by RT-q PCR and immunoprecipitation,respectively,and the expression of lncRNA Rsf1os2-encoded short peptide was detected by immunoprecipitation in macrophage polarization process and lung tissue of asthmatic mice.We then constructed overexpression lentiviral vectors for the full-length sequence of lncRNA Rsf1os2,the s ORF sequence of Rsf1os2 alone and the fulllength sequence of Rsf1os2 with mutated start codon(ATG mutated to ATT),and verified the short peptide encoded by lncRNA Rsf1os2 and the Rsf1os2 lnc RNA molecule itself by RT-PCR in macrophage polarization.By mass spectrometry and Co-IP experiments,the interacting proteins of the short peptide encoded by lncRNA Rsf1os2 were identified,and the effect of the interacting proteins of the short peptide encoded by lncRNA Rsf1os2 on macrophage polarization was determined by inhibiting the expression of their interacting proteins.Results: After validation of GFP fluorescence expression of candidate lnc RNAs by bioinformatics prediction combined with high-inclusion screening of the constructed ORFGFPmut fusion plasmid,we screened for the macrophage polarization-associated lncRNA Rsf1os2 with short peptide encoding ability.by constructing an ORF-flag fusion plasmid and verifying the expression of the short peptide by immunofluorescence and immunoprecipitation,we found that lncRNA Rsf1os2 is capable of encoding the short peptide MPM-93.lncRNA Rsf1os2 was determined to be highly expressed in M2 type macrophages by RT-q PCR.UCSC database analysis clearly localized lncRNA Rsf1os2 to mouse chromosome 7.Immunofluorescence experiments verified that the short peptide MPM-93 encoded by lncRNA Rsf1os2 was localized in the nucleus,and immunoprecipitation assays confirmed that the short peptide MPM-93 encoded by lncRNA Rsf1os2 was highly expressed in M2 macrophages and lung tissues of asthmatic mice.RT-q PCR experiments confirmed that overexpression of both Rsf1os2 and ORF promoted BMDM polarization toward M2 and inhibited M1 polarization,but ORF mutation(Rsf1os2-ORFmut)lost the above function of Rsf1os2.By mass spectrometry and Co-IP assay,Srsf-1,an interacting protein of MPM-93,was identified,and its high expression in M2 was determined by RT-q PCR;inhibition of Srsf-1 decreased the phenotype of M2 macrophages.Conclusion: The lncRNA Rsf1os2 plays an important role in allergic asthma through its encoded short peptide MPM-93 that regulates M2 macrophage polarization.