C/EBPß-Lin28a positive feedback loop triggered by C/EBPß hypomethylation enhances the proliferation and migration of vascular smooth muscle cells in restenosis.

ABSTRACT

BACKGROUND: The main cause of restenosis after percutaneous transluminal angioplasty (PTA) is the excessive proliferation and migration of vascular smooth muscle cells (VSMCs). Lin28a has been reported to play critical regulatory roles in this process. However, whether CCAAT/enhancer-binding proteins ß (C/EBPß) binds to the Lin28a promoter and drives the progression of restenosis has not been clarified. Therefore, in the present study, we aim to clarify the role of C/EBPß-Lin28a axis in restenosis. METHODS: Restenosis and atherosclerosis rat models of type 2 diabetes (n  =  20, for each group) were established by subjecting to PTA. Subsequently, the difference in DNA methylation status and expression of C/EBPß between the two groups were assessed. EdU, Transwell, and rescue assays were performed to assess the effect of C/EBPß on the proliferation and migration of VSMCs. DNA methylation status was further assessed using Methyltarget sequencing. The interaction between Lin28a and ten-eleven translocation 1 (TET1) was analysed using co-immunoprecipitation (Co-IP) assay. Student's t-test and one-way analysis of variance were used for statistical analysis. RESULTS: C/EBPß expression was upregulated and accompanied by hypomethylation of its promoter in restenosis when compared with atherosclerosis. In vitroC/EBPß overexpression facilitated the proliferation and migration of VSMCs and was associated with increased Lin28a expression. Conversely, C/EBPß knockdown resulted in the opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPß could directly bind to Lin28a promoter. Increased C/EBPß expression and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. Further, mechanical stretch promoted C/EBPß and Lin28a expression accompanied by C/EBPß hypomethylation. Additionally, Lin28a overexpression reduced C/EBPß methylation via recruiting TET1 and enhanced C/EBPß-mediated proliferation and migration of VSMCs. The opposite was noted in Lin28a knockdown cells. CONCLUSION: Our findings suggest that the C/EBPß-Lin28a axis is a driver of restenosis progression, and presents a promising therapeutic target for restenosis.