AS mouse models and MOVAS cell models were established through high-fat diet and the treatment of ox-LDL, respectively. METTL3 expression in AS models was detected via RT-qPCR and Western blot. The AS plaques, lipid deposition, and collagen fibers were examined via histological staining. The levels of Ly-6c, α-SMA, and OPN were examined via Western blot. The blood lipid indexes in mouse aortic tissues were determined using kits. The proliferation and migration of MOVAS cells were detected via CCK-8 and Transwell assays. The m6A modification level of mRNA was quantified. The binding relationship between pri-miR-375 and DGCR8, and the enrichment of m6A on pri-miR-375 were detected via RIP. The binding relationship between miR-375-3p and 3-phosphoinositide-dependent protein kinase-1 (PDK1) was verified via dual-luciferase assay. Joint experiments were designed to investigate the role of miR-375-3P/PDK1 in the phenotypic transformation of VSMCs.
METTL3 was highly expressed in AS. Silencing METTL3 alleviated AS progression and stabilized AS plaques in mice, and limited the phenotypic transformation of VSMCs induced by ox-LDL. Silencing METTL3 inhibited m6A level and decreased the binding of DGCR8 to pri-miR-375 and further limited miR-375-3p expression. miR-375-3p targeted PDK1 transcription. miR-375-3p upregulation or PDK1 downregulation facilitated the phenotypic transformation of VSMCs.
METTL3-mediated m6A modification promoted VSMC phenotype transformation and made AS plaques more vulnerable via the miR-375-3p/PDK1 axis.
方法:通过高脂饮食和ox-LDL治疗建立AS小鼠模型和MOVAS细胞模型,分别。通过RT-qPCR和Western印迹检测AS模型中的METTL3表达。AS斑块,脂质沉积,通过组织学染色检查胶原纤维。Ly-6c的水平,α-SMA,通过蛋白质印迹检查OPN。使用试剂盒测定小鼠主动脉组织中的血脂指标。通过CCK-8和Transwell实验检测MOVAS细胞的增殖和迁移。定量mRNA的m6A修饰水平。通过RIP检测pri-miR-375与DGCR8的结合关系以及pri-miR-375上m6A的富集。miR-375-3p与3-磷酸肌醇依赖性蛋白激酶-1(PDK1)之间的结合关系通过双荧光素酶测定来验证。联合实验旨在研究miR-375-3P/PDK1在VSMC表型转化中的作用。
结果:METTL3在AS中高表达。沉默METTL3可减轻小鼠AS进展并稳定AS斑块,限制了ox-LDL诱导的VSMCs的表型转化。沉默METTL3抑制m6A水平并降低DGCR8与pri-miR-375的结合并进一步限制miR-375-3p表达。miR-375-3p靶向PDK1转录。miR-375-3p上调或PDK1下调促进VSMC的表型转化。
结论:METTL3介导的m6A修饰促进了VSMC表型转化,并通过miR-375-3p/PDK1轴使AS斑块更加脆弱。