miR-10b通过激活KLF4/Notch-1/STAT3信号途径加速腹主动脉瘤的进展

MiR⁃10b accelerates the progression of abdominal aortic aneurysms by activating the KLF4/Notch⁃1/STAT3 signal pathway

目的:miR-10b促进动脉瘤的进展但其机制未明确,本实验通过检测miR-10b对KLF4/Notch-1/STAT3途径的影响阐明其机理。方法:双荧光素酶实验验证miR-10b的靶基因。取20只apoE-/-小鼠构建腹主动脉瘤模型,其中10只尾静脉注射miR-10b过表达的慢病毒,分别记为模型组和miR-10b组。另取10只apoE-/-小鼠作为假手术组,28 d后解剖小鼠腹主动脉行病理学染色、Western-blot实验。将血管紧张素Ⅱ(AngⅡ)作用后的THP-1巨噬细胞根据是否转染miR-10b mimic及其Nc对照、是否添加抑制剂分组:(1)Nc组;(2)miR-10b mimic组;(3)Nc+KLF4抑制组;(4)miR-10b mimic+KLF4抑制组;(5)Nc+Notch-1抑制组;(6)miR-10b mimic+Notch-1抑制组;(7)Nc+STAT3抑制组;(8)miR-10b mimic+STAT3抑制组,检测细胞内蛋白水平。结果:检测双荧光素酶活性得知miR-10b可靶向作用于基因KLF4。小鼠血管病理实验发现,与正常小鼠相比,动脉瘤模型小鼠的血管明显增粗、组织结构异常,miR-10b可加重血管病变;CD68+巨噬细胞、蛋白Notch-1、p-STAT3、MMP-2、MMP-9在假手术组、模型组与miR-10b逐渐增多,而α-SMA、蛋白KLF4逐渐减少。(1)、(2)两组细胞间,后者KLF4表达较少、而Notch-1、p-STAT3、MMP-2、MMP-9表达显著偏多;KLF4抑制剂抑制KLF4表达,而Notch-1显著增多;Notch-1被抑制而减少表达,p-STAT3也随之减少;p-STAT3被抑制表达,MMP-2与MMP-9表达量也明显降低。结论:miR-10b通过作用于KLF4/Notch-1/STAT3信号途径,引起动脉瘤组织中MMP-2、MMP-9表达增加,从而加速动脉瘤的进程。

Objective:miR-10b promotes the progression of aneurysms,but its mechanism is not clear.This experiment attempted to elucidate the mechanism by detecting the effect of miR-10b on KLF4/Notch-1/STAT3 pathway.Methods:Dual-luciferase assay was performed to verify the target gene of miR-10b.Twenty apoE-/-mice were taken to construct the abdominal aortic aneurysm models,10 of them were injected with mir-10b overexpressed lentivirus in the tail vein,and they were respectively designated as the model group and miR-10b group.Another 10 apoE-/-mice were used as the sham operation group,and 28 days later,the abdominal aortas of the mice were dissected for pathological staining,western-blot analysis.THP-1 macrophages stimulated by Ang II were divided into groups,according to whether they were transfected with miR-10b mimic and its negative control,and whether inhibitors were added:(1)Nc group;(2)miR-10b mimic group;(3)Nc+KLF4 inhibition group;(4)miR-10b mimic+KLF4 inhibition group;(5)Nc+Notch-1 inhibition group;(6)miR-10b mimic+Notch-1 inhibition group;(7)Nc+STAT3 inhibition group;(8)miR-10b mimic+STAT3 inhibition group,and lastly,the protein levels in the cells were then examined.Results:Detection of dual-luciferase activity showed that miR-10b could target the gene KLF4.The vascular pathology test of mice revealed that compared to normal mice,the blood vessels of aneurysm model mice were significantly thickened and the tissue structure was abnormal,and miR-10b could exacerbate vascular lesions.CD68+macrophages,proteins Notch-1,p-STAT3,MMP-2,MMP-9 increased gradually in the sham operation group,model group and miR-10b group,whileα-SMA and protein KLF4 decreased gradually.In the comparison between groups(1)and(2),the latter showed less expression of KLF4 and significantly higher expression of Notch-1,p-STAT3,MMP-2,and MMP-9;Inhibition of KLF4 led to a decrease in its expression,while Notch-1 showed a significant increase.Suppression of Notch-1 resulted in reduced expression,followed by a decrease in p-STAT3.Inhibition of p-STAT3 expression led to a significant reduction in the expression of MMP-2 and MMP-9.Conclusion:miR-10b acts on the KLF4/Notch-1/STAT3 signaling pathway to increase the expression of MMP-2 and MMP-9 in aneurysm tissue,thereby accelerating the process of aneurysm.