CHEN Wenwen,ZHANG Min,FANG Su, et al. NSUN2 mediates the activation of CD4+T cells in systemic lupus erythematosus through m5C modification[J]. JOURNAL OF WEZHOU MEDICAL UNIVERSITY, 2024, 54(8): 603-613.
Abstract：Objective: To investigate the effect of mRNA m5C methyltransferase NSUN2 on CD4+ T cell activation in patients with systemic lupus erythematosus (SLE). Methods: The NSUN2-stable knockout Jurkat cell monoclonal strain was constructed by lentivirus infection and limited dilution method, and the knockout effectiveness of NSUN2 in Jurkat cells was detected by Western blot and immunofluorescence. Dot blot analysis was made of m5C modification level after NSUN2 knockout. Jurkat cells were activated with anti-human CD3/CD28 antibodies, and the expression levels of Jurkat cell activation markers CD69 and CD25 were detected by flow cytometry at 24 h and 48 h, respectively. The transcription levels of IL-2 and TNF-α after Jurkat cell activation were detected by RT-qPCR. A mouse model of NSUN2+/- was established, CD4+ T cells were isolated by magnetic beads, and the effect of NSUN2 knockdown on CD4+ T cell activation was further evaluated by inducing cell activation in vitro. Arraystar mRNA epigenome chip was used to detect the m5C modification and gene expression level in Jurkat cells after NSUN2 knockout, and m5C modified target genes regulated by NSUN2 were obtained.Combined with the m5C modified gene set of differences in CD4+ T cells of SLE patients set that differs in CD4+T cells of SLE patients, target genes regulated by NSUN2 and associated with SLE disease were screened, and then GO and KEGG enrichment was further used to analyze the potential pathway of NSUN2 regulating CD4+
T cell function in SLE. Results: The NSUN2-knockout Jurkat cell monoclonal strain was successfully obtained using CRISPR-Cas9 technology. Compared with the NSUN2-NC group, the m5C modification level was downregulated in the NSUN2-KO group, while the expression levels of Jurkat cell activation markers CD69 and CD25 were up-regulated. The expression levels of activation-related cytokines IL-2 and TNF-α were up-regulated (P<0.05). Compared with wild-type mice, the expression level of CD69, an activation marker of Naive CD4+ T cells in NSUN2+/- mice, was up-regulated. It was found that NSUN2-regulated gene sets existed in Jurkat cells,and GO and KEGG enrichment showed that these genes were related to T cell activation. Combined with the m5C modified gene set that differs in CD4+ T cells of SLE patients, the target gene set regulated by NSUN2 and associated with SLE disease was screened. GO and KEGG enrichment analysis confirmed that these gene sets were associated with CD4+ T cell function. Conclusion: NSUN2 knockdown promotes the activation of Jurkat cells and Naive CD4+ T cells in mice, and the potential signaling pathway of NSUN2 regulation of CD4+ T cell activation in SLE patients are preliminary screened.
