Zhou Xiaojun, Jiang Shan, Guo Siyi, Yao Shuai, Sheng Qiqi, Zhang Qian, Dong Jianjun, Liao Lin
Department of Endocrinology and Metabology, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Shandong Key Laboratory of Rheumatic Disease and Translational Medicine, Shandong Institute of Nephrology, Jinan, Shandong 250014, China.
Department of Endocrinology and Metabology, Shandong Provincial Qianfoshan Hospital, Cheeloo College of Medicine, Shandong University, Jinan, Shandong 250014, China.
Chin Med J (Engl). 2025 Feb 20;138(4):419-429. doi: 10.1097/CM9.0000000000003110. Epub 2024 May 28.
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 β (C/EBPβ) 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/EBPβ-Lin28a axis in restenosis.
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/EBPβ between the two groups were assessed. EdU, Transwell, and rescue assays were performed to assess the effect of C/EBPβ 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.
C/EBPβ expression was upregulated and accompanied by hypomethylation of its promoter in restenosis when compared with atherosclerosis. In vitroC/EBPβ overexpression facilitated the proliferation and migration of VSMCs and was associated with increased Lin28a expression. Conversely, C/EBPβ knockdown resulted in the opposite effects. Chromatin immunoprecipitation assays further demonstrated that C/EBPβ could directly bind to Lin28a promoter. Increased C/EBPβ expression and enhanced proliferation and migration of VSMCs were observed after decitabine treatment. Further, mechanical stretch promoted C/EBPβ and Lin28a expression accompanied by C/EBPβ hypomethylation. Additionally, Lin28a overexpression reduced C/EBPβ methylation via recruiting TET1 and enhanced C/EBPβ-mediated proliferation and migration of VSMCs. The opposite was noted in Lin28a knockdown cells.
Our findings suggest that the C/EBPβ-Lin28a axis is a driver of restenosis progression, and presents a promising therapeutic target for restenosis.
经皮腔内血管成形术（PTA）后再狭窄的主要原因是血管平滑肌细胞（VSMC）过度增殖和迁移。据报道，Lin28a在此过程中发挥关键调节作用。然而，CCAAT/增强子结合蛋白β（C/EBPβ）是否与Lin28a启动子结合并驱动再狭窄进展尚不清楚。因此，在本研究中，我们旨在阐明C/EBPβ-Lin28a轴在再狭窄中的作用。
通过PTA建立2型糖尿病再狭窄和动脉粥样硬化大鼠模型（每组n = 20）。随后，评估两组之间DNA甲基化状态和C/EBPβ表达的差异。进行EdU、Transwell和挽救实验以评估C/EBPβ对VSMC增殖和迁移的影响。使用Methyltarget测序进一步评估DNA甲基化状态。使用免疫共沉淀（Co-IP）实验分析Lin28a与十一-易位酶1（TET1）之间的相互作用。采用学生t检验和单因素方差分析进行统计分析。
与动脉粥样硬化相比，再狭窄时C/EBPβ表达上调，其启动子伴有低甲基化。体外实验中，C/EBPβ过表达促进VSMC增殖和迁移，并与Lin28a表达增加相关。相反，C/EBPβ敲低产生相反的效果。染色质免疫沉淀实验进一步证明C/EBPβ可直接结合Lin28a启动子。地西他滨处理后观察到C/EBPβ表达增加以及VSMC增殖和迁移增强。此外，机械拉伸促进C/EBPβ和Lin28a表达，同时伴有C/EBPβ低甲基化。另外，Lin28a过表达通过招募TET1降低C/EBPβ甲基化，并增强C/EBPβ介导的VSMC增殖和迁移。在Lin28a敲低细胞中观察到相反的结果。
我们的研究结果表明，C/EBPβ-Lin28a轴是再狭窄进展的驱动因素，是再狭窄有前景的治疗靶点。
