Screening of OX40L gene related to Th17 cell function and verification of its targeted relationship with miR-146a-3p,and its regulatory mechanism on Th17 cell migration
Objective To screen the Th17 cell function-related gene OX40L,to verify its targeted relationship with microRNA 146a-3p(miR-146a-3p),and to observe its regulatory effect on Th17 cell migration.Methods ① The GSE57098 gene dataset was screened out by searching for multiple sclerosis in the GEO dataset,differential genes between CD4+T cells and Th17-like cells were obtained using R language,GO analysis and KEGG analysis were performed using DAVID 6.8 online analysis software,protein interactions were obtained from STRING online analysis website,Hub pro-teins related to Th17 cell function were obtained using Cytoscape software,and the key protein OX40L was selected and validated by qPCR.② The binding site between miR-146a-3p and OX40L was predicted by Starbase,and double lucifer-ase reporter assay was used to verify the targeted relationship between miR-146a-3p and OX40L.③ Th17 cells that grew well were cultured in vitro,and were treated with miR-146a-3p mimics,inhibitors,and controls.MiR-146a-3p was detect-ed by qRT-PCR,and the proliferation of Th17 cells was detected by CCK-8.The migration of Th17 cells was detected by Transwell,the apoptosis ratio of Th17 cells was detected by flow cytometry,qRT-PCR and Western blotting were used to detect OX40L mRNA and protein,and ELISA was used to detect the TNF-α level in each group.Results ① There were 2 130 differentially expressed genes between Th17 cells and CD4+cells in GSE57098,of which 1 515 were up-regulated and 615 were down-regulated.OX40L ranked high among up-regulated proteins,which was involved in the Cytokine Re-ceptor Interaction pathway by KEGG analysis.② MiR-146a-3p had a targeted binding relationship with OX40L mRNA3'UTR,and miR-146a-3p could inhibit the transcriptional expression of OX40L gene.③ Conclusions OX40L had significant changes in the gene expression profile of Th17 cells,and its up-regulation was one of the main mechanisms of multiple sclerosis;miR-146a-3p further inhibited the translation level of NFκB p65 in the TNF-α pathway and reduced the migration of Th17 cells by regulating the expression of OX40L.
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