| Objective:Based on bioinformatics prediction and preliminary date in our lab, the research was aimed to investigate the role of inflammatory cytokines IL-6 and TNF-? in the regulation of miR-34 a, miR-31 and their target gene Foxp3. And we want to discuss whether the downregulation of Foxp3 by these miRNAs is correlated with the effects on Foxp3~+ Treg/ROR?t+ Th17 imbalance and progression of ongoing autoimmune diseases characterized by dramatic inflammatory reaction. And consequently, our data in part represented an entry point for better understanding the mechanism of certain autoimmune diseases with an eye toward developing novel treatment strategies. Methods:Correlation between miR-34 a, miR-31 and Foxp3 expression in HEK-293 T cells was determined by bioinformatics prediction and preliminary data at first. Murine miR-31 precursor was amplified by PCR and inserted into pLV lentiviral miRNA expression vector and MGP retroviral expression vector as pLV-miR-31 lentiviral vector and MGP-31 retroviral vector using recombinant DNA technology. HEK-293 T cells were cotransfected with p LV-miR-31, MGP-31 or pSMPUW-miR-34a(constructed previously) recombinant vector plus pCDNA-mFoxp3-3'-UTR expression vector(PFU) to identify Foxp3 transcriptional and translational levels by Quantitative real-time PCR(qPCR) and Immunoblot respectively. Moreover, mouse CD4+ T cells and the mouse lymphoma cell line EL4 cells were treated with TGF-? for the induction of Foxp3 in EL4 and CD4+ T cells, the correlation between miR-34 a, miR-31 and Foxp3 was performed by qPCR. To determine whether miR-34 a and miR-31 were regulated by inflammatory cytokines, na?ve CD4+ CD62L+ T cells purified from mouse splenic cells by magnetic cell sorting were cultured with IL-1?, IL-6, IL-23, TGF-?, anti-IFN-? and anti-IL-4 to induce Th17 cells in vitro, the percentage of CD4+ IL17+ Th17 cells was indentified by intracellular staining and transcriptional levels of miR-34 a and miR-31 were detected by qPCR. The expression levels of miR-34 a and miR-31 were also detected in mouse CD4+ T cells and EL4 upon being treated with IL-6 and TNF-? respectively by qPCR, NF-?B phosphorylation and Foxp3 protein levels were detected by Flow cytometry(FCM) and Immunoblot under this condition. At last, to elaborate the mechanism of downstream signals, NF-?B signaling pathways was activated or inactivated in the inflammatory microenvironment induced by EL4 or CD4+ T cells treated with IL-6 or TNF-? along with BAY 11-7082 respectively, the expression levels of miR-34 a and miR-31 were detected by qPCR and expression level of Foxp3 was determined by Immunoblot. Results:The results of DNA sequencing showed that p LV-miR-31 lentiviral vector and MGP-31 retroviral vector were constructed successfully. The results of qPCR and immunoblot indicated that miR-34 a inhibited Foxp3 protein level and miR-31 inhibited not only Foxp3 mRNA but also protein level in HEK-293 T cells. miR-34 a, as well as miR-31, was downregulated and Foxp3 mRNA was upregulated in EL4 or mouse CD4+ T cells treated with TGF-? but miR-34 a and mIR-31 were upregulated in Th17 cells, and these results indicated that miR-34 a and miR-31 expression were negatively correlated with TGF-? induced Foxp3 but positively correlated certain Th17-skewing cytokines. Upregulation of miR-34 a, miR-31 expression and NF-?B phosphorylation were detected after CD4+ T and EL4 cells were treated with IL-6 and TNF-?, and the opposite results were observed when cells got extra treatment with BAY 11-7082. Conclusion:NF-?B was activated by certain cytokines such as IL-6 and TNF-? secreted from inflammatory microenvironment and promoted transcription of miR-34 a and miR-31 which functioned as repressors for Foxp3 expression, the IL-6/TNF-?-NF-?B-miR-34a/miR-31 axis accelerated disruptive Foxp3~+ Treg/ROR?t+ Th17 balance, thereby facilitating the progression of ongoing autoimmune diseases. |
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