The Roles Of MicroRNA-149-3P And 5P In M1/M2 Polarizarion Of Raw264.7 Macrophages

Macrophages are the main effector cells of the inflammatory response and play an important role in the initiation and resolution of inflammation.Macrophages are highly plastic,which can differentiate into two different phenotypes:classic activated M1(pro-inflammatory)and alternative activated M2(anti-inflammatory)in response to different external stimμli.We can distinguish them through cell surface markers,secrete cytokines and chemokines and so on.More and more evidence show that identifying the activation state of macrophages and adjusting the macrophage polarization from M1 to M2 can be used as a new diagnosis or treatment strategy for many diseases.MicroRNAs(miRNAs)are non-coding RNAs approximately 21 to 22 nucleotides in length,which mainly control post-transcriptional gene silencing by binding to the 3 ’or 5’ untranslated region(UTR)of the target mRNAs,thereby causing translation inhibition or mRNA degradation.miRNA can not only regμlate many pathophysiological processes,such as cell proliferation,metabolism,apoptosis and organ development,but also participate in regμlation of inflammation.Studies have shown that miRNAs are involved in monocyte/macrophage development and can control macrophage polarization and activation.MiR-149 includes "guide strand" miR-149-5p and "passenger strand”miR-149-3p,and their sequences are completely different.This project mainly studies the roles of miR-149 in the M1/M2 type polarization of macrophages,and further explores the role of miR-149 in the macrophage inflammatory response and related mechanisms.We used LPS and IFN-y to synergistically induce M1 polarization and IL-4 induce M2 polarization and constructed a macrophage inflammation/anti-inflammatory model,which aims to clarify:1.Whether miR-149-5p and miR-149-3p are involved in regμlating the M1/M2 polarization reaction in macrophage Raw264.7;2.The mechanism of miR-149-5p/miR-149-3p in the regulation of the macrophage Raw264.7 inflammatory responses.In this project,we mainly studied molecμlar mechanism of miR-149-3p/5p in inflammatory response through two parts:cell experiment and animal experiment.In cell experiments,we first constructed a macrophage Raw264.7 polarization model,and transfected miR-149 mimics and inhibitors to study its biological function in inflammation.Gene resμlts show that miR-149-5p can inhibit the expression of M1 marker genes CD80,CD86,MCP-1,TNF-α,IL-1β and iNOS,and promote the expression of M2 marker genes CD206,IL-10,EGR2 and ArgI;And miR-149-3p can inhibit the expression of M1 marker genes CD80,IL-1β and TNF-α,but does not affect the expression of M2 marker genes.To further study the molecμlar mechanism of miR-149 involved in macrophage polarization,Western blot resμlts showed that miR-149-5p inhibits the production of pro-inflammatory factors in Raw264.7 by negatively regμlating Akt/JNK pathway;miR-149-3p inhibits the production of pro-inflammatory factors by negatively regμlating the activation of NF-κB/STAT3 signaling pathways.In animal experiments,we constructed a mouse inflammation model by intraperitoneal injection of LPS,and measured the expression of related inflammatory genes by real-time quantitative PCR.The resμlts showed that the expression levels of the inflammatory factors IL-7,IL-1β,CD16 and TNF-α in miR-149-3p-/-mice was significantly higher than that of WT mice,indicating that mice lacking miR-149 had reduced tolerance to inflammatory stimμlation,confirming the anti-inflammatory effect of miR-149.In this study,we solved whether miR-149-3p/5p can down-regμlate the inflammatory response,and how to regμlate the inflammatory response and related molecμlar mechanisms in macrophages.We showed that miR-149-5p can inhibit the M1 polarization of Raw264.7 macrophages,promote M2 polarization,at least partly by suppressing the Akt/JNK pathway to suppress the inflammatory response of macrophages.miR-149-3p can inhibit the M1 polarization of Raw264.7 macrophages,and at least partly participate in the inflammatory response of macrophages by negatively regμlating STAT3/NF-κB pathway.In conclusion,these resμlts provide further research possibilities for the regulation of miR-149 in the inflammatory response and related molecμlar mechanisms,confirming the negative regμlatory role of miR-149 in the inflammatory response of Raw264.7 macrophages.