| PartⅠ MicroRNA-155expression correlated with Th cell differentiation in patients with coronary heart diseaseObjectives:To investigate the levels of inflammation-related miRNAs and the proportions of Th cell subsets in peripheral blood of patients with CHD, and the relationship between them.Methods:Patients with SA, UA and AMI were collected as study subjects and patients with CPS were collected as controls, and every group including14-15patients. Peripheral blood was collected, plasma and PBMCs were obtained after centrifugation and Ficoll-Hypaque density gradient centrifugation, respectively. RNA samples from plasma and PBMCs were extracted by Trizol LS and Trizol, respectively, and the inflammation-related miRNAs levels were detected by qRT-PCR; the proportions of Th cell subsets were detected by flow cytometric analysis; and the distribution of miR-155and IL-17A in cells were detected by FISH and immunohistochemical, respectively.Results:Compare with their levels in PBMCs of patients with CPS, miR-21and miR-146a levels were higher and miR-155level was lower in PBMCs of patients with UA and AMI (p<0.01), while there were no significant differences in the expression of miR-125a-5p, miR-125b and miR-31among the groups (p>0.05). In plasma, the expression patterns of miRNAs were in accordance with their patterns in PBMCs, except for miR-21. MiR-21level in plasma of patients with AMI were6-fold higher than its level in plasma of patients with CPS (p<0.01), but there was no significant difference between patients with UA and with CPS (p>0.05). No significant differences were observed for the levels of miRNAs between patients with SA and with CPS (p>0.05). Furthermore, we detected the proportions of Th cell subsets in PBMCs of patients with CHD. The results showed that the proportions of Thl and Th17cells were increased (p<0.01), the proportion of Treg cells was decreased (p<0.01), whereas the proportion of Th2cells had no significant changes (p>0.05) in PBMCs of patients with UA and AMI compare with those in patients with CPS. Moreover, the Spearman’s correlation analysis showed that the miR-155level in PBMCs was highly inversely correlated with the frequency of Th17cells in patients with AMI and UA (r=-0.896, p<0.01). And miR-155and IL-17A co-expressed in the same cells in patients with ACS.Conclusion:Inflammation-related miRNAs specifically expressed in peripheral blood of patients with ACS and miR-155expression correlated with the imbalance of Th cell differentiation. Part Ⅱ The mechanisms involved in microRNA-155inducing CD4+T cells differentiationObjectives:To clarity how miR-155regulates CD4+T cell subsets (including Thl, Th2, Th17, and Treg cells) differentiation and function. And to detect the target and signaling pathways involved in these process.Methods:Spleens were dissected from BALB/c mice, then splenic mononuclear cells were obtained by grind and Ficoll-Hypaque density gradient centrifugation; CD4+T cells were purified by CD4+T Cell Isolation Kit Ⅱ (the purity>95%); oligonucleotides (including pre-miR-155, pre-miR-ctrl, anti-miR-155, anti-miR-ctrl, SOCSl-TPmiR-155, and control-TPmiR-155) were transfected into CD4+T Cell by nucleofection; CD4+T cells were activated and polarized by specific antibodies; the proportions of Th cell subsets were detected by flow cytometric analysis; the mRNA levels of transcriptional regulators were detected by qRT-PCR; the mRNA and protein levels of cytokines were detected by qRT-PCR and ELISA, respectively; the transcripts levels in the signaling pathways were detected by western boltting.Results:The proportions of Thl7and Treg cells and their specific transcriptional regulators, ROR-yt and Foxp3, were up-regulated when miR-155was over-expressed (p<0.05). In contrast, they were down-regulated when miR-155was inhibited (p<0.05). Moreover, the functional cytokines of Th17cells, IL-17A, was also up-regulated by over-expressed miR-155and down-regulated by inhibited miR-155(p<0.05), but the functional cytokines of Treg cells, IL-10and TGF-β1, were regulated neither by over-expressed nor by inhibited miR-155(p>0.05). Further study showed that the feedback regulator of JAK/STAT signaling pathway, SOCS1, was the direct target of miR-155. During CD4+T cell differentiation, miR-155positively regulated JAK/STAT signaling pathway by directly inhibiting the expression of SOCS1, which in tune up-regulated the differentiation of Th17and Treg cells, and the function of Th17cells. Furthermore, we confirmed that miR-155positively regulated the expression of the functional transcriptional regulators and cytokines of Th1, T-bet and IFN-y, and negatively regulated the expression of the functional transcriptional regulators and cytokines of Th2, GATA3and IL-4(p<0.05).Conclusion:MiR-155positively regulated the differentiation and function of pro-inflammatory CD4+T cells, Thl and Th17cells, whereas negatively regulated the differentiation and function of anti-inflammatory CD4+T cells, Th2cells. Moreover, miR-155also positively regulated the differentiation of Treg, but not the function of it. In conclusion, miR-155induced the imbalance of CD4+T cells differentiation. Part IIIMicroRNA-155induces RAW264.7cells differentiation into DCsObjectives:To clarify how miR-155regulates the expression of key surface molecules and cytokines by RAW264.7cells.Methods:RAW264.7cells were cultured in DMEM medium+10%fetal bovine serum (FBS); oligonucleotides (including pre-miR-155, pre-miR-ctrl, anti-miR-155and anti-miR-ctrl) were transfected into RAW264.7cells by Attractene Transfection Reagent; cells were stimulated by LPS and oxLDL, respectively; miR-155level was detected by qRT-PCR; surface molecules levels were detected by flow cytometric analysis; the mRNA and protein levels of cytokines were detected by qRT-PCR and ELISA, respectively.Results:The results showed that miR-155level was increased by LPS for about200-fold (p<0.01) and by oxLDL for about2-fold (p<0.05). Furthermore, the gain-and loss-of-function study showed that miR-155induced the expression of surface molecules (including MHC-II, MHC-I, CD86, and CD83) and the production of pro-inflammatory cytokines (including IL-12, IL-6, and IL-lb) both in LPS-and in oxLDL-treated RAW264.7cells (p<0.05). Additionally, miR-155also induced the expression of CD36in oxLDL-treated RAW264.7cells (p<0.01).Conclusion:Up-regulated miR-155is able to enhance the expression of surface molecules and the production of pro-inflammatory cytokines both in LPS-and in oxLDL-treated RAW264.7cells, which are neccassy for DCs maturation and antigen-presenting function of it. These results suggest that miR-155is one important factor that involves in inducing RAW264.7cells convert into DC-like cells, which is critical for the progression of immune responses as well as AS. |
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