The Function And Mechanism Of MiR-130b In The Activation Of CD4~+ T Cell

CD4~+T cells are crucial components of cellular immunity,with their immune functionality primarily reliant on activation.Alongside TCR-MHC,costimulatory molecules,and cytokines,various signal molecules and transcription factors within T cells significantly contribute to the activation process of CD4~+T cells.micro RNAs(miRNAs)are a class of non-coding RNA molecules that regulate gene expression by degrading or inhibiting the translation of target m RNAs.As a result,they play crucial roles in cellular stability,proliferation,and pathogenesis.Limited research suggests that miRNAs may also be involved in T cell activation.In this current study,a microarray analysis was conducted to investigate the expression profiles of miRNAs in DP and SP T cells.The findings revealed that the expression of miR-130b was significantly higher in DP cells compared to SP cells,and this result was further confirmed through quantitative polymerase chain reaction(Q-PCR).In vitro stimulation of splenic CD4~+T cells from WT mice resulted in a significant decrease in miR-130b expression as the duration of stimulation increased.This finding implies that the downregulation of miR-130b expression in activated CD4~+T cells may play a role in maintaining T cell survival equilibrium and preventing excessive T cell activation.Consequently,our findings suggest a potential influence of miR-130b on T cell activation.To initially investigate the role of miR-130b in T cells,a chimeric mouse model with overexpression of miR-130b was constructed.Phenotypic analysis revealed a significant reduction in the population of DP and SP cells within the miR-130b-overexpressing cell population(GFP positive cells)in the thymus,as compared to the wild-type cell population(GFP negative cells).Furthermore,in the spleen,there was a decrease in the proportion of CD4~+and CD8~+T cells within the miR-130b-overexpressed cell population,and this decrease was positively correlated with the level of miR-130b expression.In comparison to WT cell population,the experimental group exhibited a notable decrease in the proportions of naive T cells within the CD4~+and CD8~+T cell populations in the spleen.Conversely,there was a significant increase in the proportion of activated T cells.These findings suggest that the overexpression of miR-130b may induce spontaneous activation of T cells.To further investigate the effect of miR-130b on T cell development and activation,we constructed miR-130b knockout mice.There was no significant change in the proportion of T cell subsets in thymus and spleen in the knockout mice;the activation status,The proliferation and apoptosis of naive T cells in the spleen were also not significant variety.However,when splenic CD4~+T cells of WT and miR-130b KO mice were activated in vitro,the cell proliferation level of the knockout group was significantly lower than that of the WT group;the expression level of related cytokines was also significantly lower than that of WT.These results exhibited that the deficiency of miR-130b can inhibit the activation of CD4~+T cells to a certain extent.At the same time,we also established miR-130b transgenic(TG)mouse model to prove that miR-130b does promote CD4~+T cell activation.Compared with WT,the proportion of DP and SP cells in the thymus of miR-130b TG mice did not change significantly.In the spleen,the proportion of CD4~+and CD8~+T cells in miR-130b TG mice were decreased significantly;the proportion of naive T cells in the CD4~+and CD8~+T cell populations were decreased significantly,and the proportion of activated T cells were increased accordingly.This further indicated that overexpression of miR-130b caused spontaneous activation of T cells.The proliferation experiment results showed that when CD4~+T cells were stimulated with CD3 plus CD28 antibody,the proliferation level of the experimental group was significantly higher than that of the WT group;the expression level of related cytokines was also significantly higher than that of the WT group.In the resting state,there was no significant change in the apoptosis of CD4~+T cells in the spleen,but the apoptosis of CD4~+T cells in miR-130b TG mice was increased significantly after activation.This suggests that under activation conditions,when miR-130b is overexpressed,it may cause activation-induced apoptosis.The above data showed that miR-130b did promote the activation and proliferation of CD4~+T cells.How does miR-130b affect T cell activation?Through bioinformatics analysis and literature review,we selected PTEN and FOXO1 as two regulatory molecules for T cell activation.Using Western-blot and Q-PCR,we initially deduced that FOXO1 is likely a potential target gene of miR-130b.Then luciferase reporter asssay were conducted to identify FOXO1 as a potential target gene of miR-130b.To explore how miR-130b affects T cell activation and proliferation through FOXO1,we examined the expression of T cell-related activation signaling molecules.The results showed that although the total ERK and AKT were not changed,the phosphorylated ERK and AKT were decreased in miR-130b KO and increased in miR-130b TG CD4~+T cells.This suggests that miR-130b is likely to target FOXO1 and then affect the activation and proliferation of CD4~+T cells through regulateing the phosphorylation of ERK or AKT.In summary,we successfully constructed miR-130b chimeric,TG,and KO mice models;proved that miR-130b promotes the activation of CD4~+T cells,the transcription factor FOXO1 is a potential target gene of miR-130b,miR-130b promotes the activation and proliferation of CD4~+T cells by targeting FOXO1 and then through regulating the phosphorylation of ERK or AKT to involve into the activation of T cells.