C/EBPβ-Lin28a轴在糖尿病下肢血管病变球扩后再狭窄中的作用机制研究

Percutaneous transluminal angioplasty (PTA) has currently become an effective measure to lower extremity arterial disease in diabetes. However, high incidence of restenosis after PTA severely affects the prognosis of patients. Our previous studies have proved that vascular smooth muscle cells (VSMCs) migration and proliferation were the leading cause of restenosis and targeted intervention of Lin28a significantly inhibited restenosis formation, whereas the underlying mechanism for Lin28a overexpression remains unclear. Recent research showed C/EBPβ was involved in vascular remodeling and promoted VSMCs migration and proliferation. Furthermore, researches have confirmed that increased C/EBPβ expression can be mediated by demethylation of C/EBPβ. Then we further demonstrated that C/EBPβ worked as a significant upstream of Lin28a and methylation of C/EBPβ was decreased in restenosis accompanied by the elevated expression of C/EBPβ. Combined with the latest finding that Lin28a is also involved in promoting demethylation of C/EBPβ, we speculated that after PTA, occurrence of C/EBPβ activation up-regulates Lin28a, then forming a positive feedback loop to amplify C/EBPβ promoting Lin28a persistent overexpression effect, and eventually leading to excessive migration and proliferation of VSMCs. Therefore, the present study is designed to explore the role of C/EBPβ demethylation and specific mechanism to regulate Lin28a in restenosis by bisulfite modified cloning and sequencing, luciferase reporter assay and other methods in vivo and in vitro, which may provide potential molecule targets and a novel idea for prevention and treatment of PTA postoperative restenosis from epigenetic perspective.

再狭窄（RS）严重影响球囊扩张术（PTA）后糖尿病下肢血管病变患者预后，血管平滑肌细胞（VSMCs）迁移增殖是RS发生的主因。前期研究示Lin28a持续高表达是再狭窄中VSMCs迁移增殖的关键，但介导其增高的机制不明。近期研究示上调C/EBPβ可促VSMCs迁移增殖，参与血管重构，且C/EBPβ表达受其DNA甲基化影响明显。通过预测并验证C/EBPβ是Lin28a潜在上游因子，检测发现RS处C/EBPβ甲基化降低伴随蛋白升高。结合新近研究Lin28a可在C/EBPβ启动子区富集，促C/EBPβ去甲基化，我们推测：C/EBPβ在RS中被激活后上调Lin28a，并通过两者间的正反馈作用，促VSMCs过度迁移增殖，致RS快速进展。本课题拟利用亚硫酸氢盐修饰后克隆测序、荧光素酶报告基因检测等方法，体内体外逐层验证C/EBPβ-Lin28a轴在RS发生中的作用，从表观遗传学角度为防治RS提供新靶点。

背景：球囊扩张术后再狭窄的主要原因是血管平滑肌细胞的过度迁移增殖，转录因子全称（C/EBPβ）在血管重塑过程中发挥重要作用，然而C/EBPβ在再狭窄中的作用尚未阐明。.方法：我们通过建立2型糖尿病再狭窄与动脉粥样硬化大鼠模型来检测C/EBPβ的表达及甲基化水平在再狭窄与动脉粥样硬化中的差异，同时体外通过调控C/EBPβ和Lin28a来明确对血管平滑肌细胞增殖迁移的作用，并通过用去甲基化药物地西他滨明确DNA甲基化对C/EBPβ和Lin28a表达的影响以及进一步对血管平滑肌细胞增殖迁移的调控作用。.结果：与AS相比，C/EBPβ在RS中高表达且甲基化水平较低。过表达C/EBPβ，VSMCs增殖迁移增多，且Lin28a表达增加，干扰C/EBPβ结果相反。ChIP实验进一步证实C/EBPβ和与Lin28a启动子结合。同时Lin28a过表达组C/EBPβ表达增加，Lin28a干扰组结果相反。地西他滨处理后，C/EBPβ表达增加，同时VSMCs增殖迁移增加。为模拟体内球囊扩张对细胞的作用，对细胞进行牵张处理后C/EBPβ和Lin28a表达增加。.结论：与AS相比，C/EBPβ在RS中低甲基化高表达，受甲基化调控，同时作为转录因子结合Lin28a启动子上调Lin28a表达，并通过两者间的正反馈环路，促进VSMCs增殖迁移。我们的研究表明 C/EBPβ-Lin28a 轴是再狭窄进展的驱动因素，为再狭窄提供了新的治疗靶点。