CCL20/CCR6 has recently been shown to play vital roles in the development of atherosclerosis, yet few reports have described the mechanisms involved. Here, we explore detailed molecular mechanisms of the involvement of the CCL20/CCR6 axis in atherogenesis.Design and Method:
In our study, CCR6−/−ApoE−/− and ApoE−/− mice were employed and divided into diet-induced atherosclerosis model group. UBM and H&E stain were used to evaluate atherosclerotic plaque size. Immunohistochemistry and RT-PCR analysis were used to assess CCR6 and CCL20 expression. 760 miRNAs in mouse vessels were screened. Potential miRNA target sites were predicted by computer software and verified with the Dual Luciferase Reporter assay system. Mouse aortic endothelial cells were transfected and tube formation assay was performed.Results:
Results showed a significant reduction in plaque size and the expressions of CCR6 /CCL20 and macrophages accumulation in CCR6−/−ApoE−/− mice compared with ApoE−/− mice. Screening of 760 miRNAs found 10 miRNAs with fold changes (2−ΔΔCT) > 3.0-fold, RT-PCR re-determined that miRNA-27b expression had the greatest fold-change. Potential miRNA-27b target sites were analyzed by predictive computer software, Naa15, a gene closely associated with neovascularization, was identified, further verified with the Dual Luciferase Reporter assay in NIH3T3 cell lines. In addition, immunohistochemistry and RT-PCR analysis revealed a substantial increased in Naa15 expression in the vascular tissues in CCR6−/−ApoE−/− mice compared to ApoE−/− mice. Interestingly, immunofluorescence staining showed a significant decreased in CD31 expression in the atherosclerotic plaques of CCR6−/−ApoE−/− mice. Also, transfection of siRNA Tbdn-1/siRNA NC or miRNA27b- mimic/miRNA-NC into mouse aortic endothelial cells caused significant increased in tube formation compared to control.Conclusions:
CCR6 deletion effectively ameliorates atherosclerosis progression, potentially through inflammation or its regulatory effects on the differential expression of miRNA-27b and its target gene Tbdn-1, which in turn modulates plaque angiogenesis, and ultimately improves plaque process including area and stability.