Nf1基因沉默对小鼠主动脉血管平滑肌细胞增殖与迁移及表型转化的影响

Effect of Nf1 gene silencing on proliferation and migration and phenotypic transformation of mouse aortic vascular smooth muscle cells

摘要:

目的]探究Nf1基因沉默对平滑肌细胞增殖和迁移的作用及可能的机制。 [方法]以小鼠主动脉血管平滑肌细胞(MOVAS)为研究对象,用Nf1 siRNA转染MOVAS后,RT-qPCR验证Nf1 siRNA转染MOVAS效率,实验分为对照组、siRNA NC组和Nf1 siRNA组。采用Ki-67免疫荧光实验检测MOVAS增殖,采用划痕实验、Transwell实验评估Nf1基因沉默对MOVAS迁移能力的影响,采用Western blot检测Nf1基因沉默对MOVAS表型标志蛋白表达水平的影响以及Ras下游信号胞外信号调节激酶(ERK)、p-ERK、蛋白激酶B(Akt)、p-Akt表达水平的差异。 [结果]与对照组相比,Nf1 siRNA组MOVAS增殖能力显著升高(升高33.23%,P＜0.05)、迁移能力显著增强(划痕实验显示增强35.47%,Transwell实验显示增强39.33%,P＜0.05或P＜0.01),收缩型蛋白平滑肌22α蛋白(SM22α)、α-平滑肌肌动蛋白(α-SMA)和钙调蛋白1(CNN1)的表达降低(分别降低18.91%、23.38%和25.08%,P＜0.05或P＜0.01),合成型蛋白骨桥蛋白(OPN)表达增加(增加58.70%,P＜0.05),p-ERK、p-Akt水平显著增加(分别增加33.30%和2.42倍,P＜0.05或P＜0.01)。 [结论]Nf1基因沉默使MOVAS增殖与迁移能力显著增强,并由收缩型向合成型的表型转化。

Abstract:

Aim To explore the effect of Nf1 gene silencing on smooth muscle cell proliferation and migration and its possible molecular mechanism. Methods Using mouse aortic vascular smooth muscle cells (MOVAS) as the research object, the efficiency of Nf1 siRNA transfection in MOVAS was verified by RT-qPCR after transfection with Nf1 siRNA. The experiment was divided into control group, siRNA NC group, and Nf1 siRNA group. Ki-67 immunofluorescence assay was used to detect the proliferation of MOVAS. Scratch assay and Transwell assay were used to evaluate the effect in the migration ability of MOVAS caused by Nf1 gene silencing. Western blot was used to detect the effect of Nf1 gene silencing on the expression levels of MOVAS phenotype marker proteins and the differences in the expression levels of downstream Ras signals extracellular signal-regulated kinase (ERK), p-ERK, protein kinase B (Akt), and p-Akt. Results Compared with the control group, the proliferation ability of MOVAS in the Nf1 siRNA group was significantly increased (increased by 33.23%, P＜0.05), migration ability was significantly enhanced (scratch assay showed an increase of 35.47%, Transwell assay showed an increase of 39.33%, P＜0.05 or P＜0.01), and the expression of contractile proteins smooth muscle 22α (SM22α), α-smooth muscle actin (α-SMA), and calmodulin 1 (CNN1) was reduced (decreased by 18.91%, 23.38% and 25.08%, P＜0.05 or P＜0.01, respectively). The expression of synthetic protein osteopontin (OPN) was increased (increased by 58.70%, P＜0.05), and the levels of p-ERK and p-Akt were significantly increased (increased by 33.30% and 2.42 times, respectively, P＜0.05 or P＜0.01). Conclusion Nf1 gene silencing can significantly enhance the proliferation and migration ability of MOVAS, and transform from a contractile phenotype to a synthetic phenotype.