The Role And Mechanism Of LncRNA U90926-encoded Micropeptide Regulating Macrophage Polarization In Viral Myocarditis

Objective: To screen and identify macrophage polarization-related lncRNAs with short peptide encoding ability;to clarify the function of lncRNA(long non-coding RNA,lncRNA)U90926 and its encoded short peptide MPM-87(MPM-87aa)in macrophage polarization,and to elucidate the mechanism of MPM-87 regulation of macrophage polarization in viral myocarditis.Methods: To identify and predict the coding potential of small open reading frames(s ORF)by bioinformatics Open reading frame of lncRNAs differentially expressed in macrophages;The Fusion Expression Plasmid orf-GFP-mut(start codon-mutated GFP,GFP-MUT)was constructed and transfected into HEK293 T cells.High content screening(HCS)technique was used to detect the expression of GFP fluorescence in the transfected cells and to screen lncRNAs with coding potential.Based on the screening results,the high coding potential lncRNA U90926 was initially targeted for further functional exploration.The expression and function of lncRNA U90926 in the process of macrophage polarization were detected by q RT-PCR,and the expression of lncRNA U90926 in the heart of CVB3-induced viral myocarditis mouse model was detected.The ORF-Flag tag fusion plasmid of lncRNA U90926 was constructed and the lentiviral expression vector was packaged.The expression of lncRNA coding short peptide was determined by using Flag antibody,western blot was used to detect the endogenous expression of MPM-87 in the process of macrophage polarization,and immunofluorescence and immunoprecipitation were used to determine the localization of MPM-87 in macrophages The expression of MPM-87 in the heart of normal and CVB3 infected mice was detected A lentiviral expression vector containing the full-length ORF sequence of MPM-87(U90926),the full-length ORF sequence of U90926(U90926-ORFMUT)of MPM-87 and the full-length ORF Start codon of U90926(ATG mutant to ATT)of Mpm-87 was constructed and used to infect RAW264.7macrophages,the role of MPM-87 in macrophage polarization was detected by q RT-PCR,Flow cytometry and bactericidal assay,and the interaction proteins of MPM-87 were screened by co-immunoprecipitation and mass spectrometry.Results: The bioinformatics software Mi Pepid(https://github.com/Mind AI/Mi Pepid),a machine learning-based micro-peptide identification tool,was applied to identify small open reading frame s ORFs and predict the coding potential of the differentially expressed lncRNAs sequences in the annotated macrophages;by setting the threshold value of coding potential(Based on the screening results,lncRNA U90926 with high coding potential was selected for subsequent functional investigation.q RT-PCR results showed that lncRNA U90926 was up-regulated in M1 macrophages and si RNA After silencing the expression of lncRNA U90926,the expression of markers in M1 macrophages was detected using q RT-PCR,and the expression of i NOS,TNF-α,and IL-12 was found to be significantly reduced,suggesting that lncRNA U90926 molecule can promote M1 polarization in macrophages.By analyzing the sequence of lncRNA U90926,we found a small open reading frame s ORF of 264 nt,which could encode a micropeptide of 87 amino acids,and tentatively named the encoded micropeptide as MPM-87(macrophage polarization micropeptide-87 aa,MPM-87).The fusion plasmid lentiviral expression vector infected with RAW264.7 of the constructed ORF sequence of MPM-87(ORF),the full-length sequence of U90926(U90926)and the full-length sequence of U90926(U90926-ORFmut)with mutation of the ORF start codon of MPM-87(ATG mutation to ATT)were detected using Flag antibody.macrophages,it was determined that only the ORF sequence of MPM-87(ORF),the full-length sequence of U90926(U90926)group could express FLAG fusion protein,while the control group and the full-length sequence of U90926(U90926-ORFmut)with mutation of the ORF start codon(ATG mutation to ATT)could not express FLAG fusion protein.Using the prepared anti-MPM-87 multiple antibodies,the expression of the micropeptide MPM-87 in M0 and M1 cells was detected by immunofluorescence in the RAW264.7 macrophage cell line,and the results showed that MPM-87 was expressed in both the cytoplasm and nucleus of macrophage cells,mainly in the cytoplasm;immunoblotting verified that the expression of MPM-87 was significantly higher in M1 macrophages than in M0 macrophages.In addition,we determined that MPM-87 expression was significantly higher in cardiac infiltrated macrophages of VM mice compared with normal mice.The fusion plasmid lentivirus of the ORF sequence of MPM-87(ORF),the full-length sequence of U90926(U90926)and the full-length sequence of U90926(U90926-ORFmut)with mutation of the ORF start codon of MPM-87(ATG mutation to ATT)were constructed and detected by q PT-PCR,flow cytometry,and E.coli sterilization assay expression vector-infected RAW264.7macrophages in M1 polarization,and found that the ORF sequence of MPM-87(ORF)and the full-length sequence of U90926(U90926)both promoted the polarization of macrophages to M1 and inhibited the polarization of macrophages to M2,but the control and the ORF start codon mutation(ATG mutation to ATT)of U90926 full-length sequence(U90926-ORFmut)group had no effect;identified by immunoprecipitation and mass spectrometry analysis,the preliminary screening identified the reciprocal protein of MPM-87.Conclusion: lncRNA U90926 has the ability to encode a short peptide;lncRNA U90926 and its encoded short peptide MPM-87 promote macrophage polarization toward M1.