| Objective:In order to clarify the role of m6 A modification in colonic mucosal macrophages participating in the progression of inflammatory bowel disease(IBD)and explore the molecular mechanism of human umbilical cord mesenchymal stem cells-derived exosomes(hucMSC-Ex)alleviating IBD by regulating m6 A modification in macrophages,providing new ideas and scientific basis for clinical application of hucMSC-Ex in the prevention and treatment of IBD.Methods:(1)Human umbilical cord mesenchymal stem cells(hucMSCs)were isolated and cultured,and hucMSC-Ex was extracted by ultra-centrifugation and identified by transmission electron microscopy,Western-blot,nanoparticle tracking analysis.(2)Mouse mononuclear macrophage leukemia cell line RAW264.7 was treated with lipopolysaccharide(LPS)to induce inflammation.After hucMSC-Ex treatment,qRT-PCR,Western-blot,flow cytometry and immunofluorescence(IF)were used to detect the expression of macrophage-related inflammatory cytokines(IL-1β,IL-6,TNF-α,IL-10),functional genes(iNOS,MCP-1,Arg1)and phenotypic markers(CD86,CD206).(3)Expression of N6 methyladenosine(m6A)modification regulators within RAW264.7was examined by qRT-PCR,Western-blot and IF,and m6 A level were measured by the m6 A RNA methylation quantitative assay kit.Meanwhile the changes in human monocytic leukemia cell line THP-1 and human colon tissues were verified with same methods.(4)The mice model of hucMSC-Ex alleviating DSS-induced IBD was constructed to detect the expression of colonic mucosal tissue associated inflammatory factors(IL-1β,IL-6,TNF-α,IL-10)and functional gene(iNOS,MCP-1,Arg1)of phenotype,then further validating the expression changes of m6 A modification regulators in colon tissue and the overall m6 A level in colonic mucosa.(5)Methylated RNA immunoprecipitation with next generation sequencing(MeRIP-Seq)was used to screen target molecules with significant changes in m6 A modification level and RNA content in colonic mucosa of mice between hucMSC-Ex group and DSS-induced IBD group,then their expressions in cells and animal models were detected by qRT-PCR,Westernblot and immunohistochemical staining(IHC).The effect of m6 A modification on the decay rate of target molecules was determined by mRNA stability detection.MeRIP-q PCR,RNA coimmunoprecipitation(RIP)and double luciferase reporter assay were used to determine the binding sites of hucMSC-Ex on target molecules.(6)RAW264.7 cells were transfected with METTL3 si RNA to explore whether hucMSC-Ex could still regulate m6 A modification to alleviate inflammation after METTL3 knockdown,and to determine the regulatory effect of hucMSC-Ex on METTL3.Results:(1)Huc MSC-Ex was successfully extracted,and the particle size met the requirements.Observation by transmission electron microscopy showed that hucMSC-Ex presented a concave circular vesicle structure.Western-blot results showed that the extracted hucMSC-Ex expressed label proteins CD81,CD9,HSP70,Alix and did not express Calnexin.(2)After hucMSC-Ex treatment,the proinflammatory cytokines IL-1β,IL-6 and TNF-αwere significantly down-regulated,the anti-inflammatory cytokine IL-10 was up-regulated,the expressions of M1-type macrophage related genes,iNOS and MCP-1,were decreased,the proportion of cells expressing CD86 was slightly decreased,but the expression of M2-type functional gene Arg1 was increased.Meanwhile,the proportion of cells expressing CD206 increased significantly,indicating that hucMSC-Ex promoted macrophage towards M2-type in the process of alleviating inflammation.(3)LPS stimulation of RAW264.7 cells caused the down-regulation of the expression of m6 A "writer" METTL3 and "reader" YTHDF1,while hucMSC-Ex treatment inhibited the downregulation trend and increased the intracellular m6 A level,which was consistent with the changes in THP-1 and clinical colon tissue.(4)Percentage of body weight,DAI,colorectal length/weight ratio,expression of inflammatory factors,and H&E staining results all showed that hucMSC-Ex effectively alleviated DSS induced IBD in mice.The results of decreased expression of M1-type cell functional genes iNOS,MCP-1 and increased expression of M2-type marker Arg1 revealed that hucMSC-Ex were involved in modulating the functional polarization of colonic mucosal macrophages in IBD.What’s more,hucMSC-Ex up-regulated the expression of METTL3 and YTHDF1,and promoted the m6 A modification of colonic mucosa.(5)Slc37a2/Serpinb1 c were identified as the target molecules for hucMSC-Ex regulation of m6 A modification in IBD mice by MeRIP-Seq.qRT-PCR,Western-blot and IHC were used to verify the expression changes of the target molecule in vivo and in vitro.The results showed that hucMSC-Ex enhanced the mRNA stability to increase the expression of Slc37a2/Serpinb1 c by upregulating its m6 A level.Visualization analysis showed that Slc37a2 showed high m6 A modification in the 3’UTR region,while Serpinb1 c showed high m6 A modification peak in the CDS region,which may be the target site of hucMSC-Ex regulation of METTL3.The combination sites of METTL3 on the 3’UTR of Slc37a2 and the CDS of Serpinb1 c were identified by MeRIP-q PCR,RIP and double luciferin reporter assay.(6)After METTL3 knockdown in RAW264.7 cells,qRT-PCR,Western-blot,flow cytometry and IF results all showed that hucMSC-Ex couldn’t mediate phenotype polarization to relieve inflammation,and the expression levels of METTL3 and YTHDF1 were no longer affected by hucMSC-Ex.Meanwhile,the decay rate of Slc37a2/Serpinb1 c mRNA was accelerated and there was no significant change with the addition of hucMSC-Ex.The results of MeRIP-q PCR also showed that the m6 A modified level of Slc37a2 in the hucMSC-Ex group was not significantly different from that in the LPS group,which suggested that hucMSC-Ex increase the m6 A level of Slc37a2/Serpinb1 c to relieve macrophage inflammation by regulating METTL3.Conclusion:Huc MSC-Ex alleviated IBD by upregulating the m6 A level of Slc37a2/Serpinb1 c in the colonic mucosal macrophage to mediate phenotype transition via the METTL3-Slc37a2/Serpinb1c-YTHDF1 axis. |
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