CIRBP对小鼠血管平滑肌细胞增殖及迁移的调控及机制研究

Regulatory effect of cold-inducible RNA-binding protein on proliferation and migration of mouse vascular smooth muscle cells and its mechanism

目的:探讨冷诱导RNA结合蛋白(CIRBP)对小鼠血管平滑肌细胞(VSMCs)增殖及迁移过程的调控及机制。方法:将雄性C57BL/6J小鼠分为假手术对照组、假手术干扰组、颈总动脉损伤对照组和颈总动脉损伤干扰组,每组5只。造模后按照组别分别用阴性对照腺病毒(AD-NC)和沉默CIRBP腺病毒(AD-CIRBPI)转染,28 d后观察CIRBP表达及血管内膜的增生情况。将小鼠VSMCs分为对照组和腺病毒沉默组,分别用AD-NC和AD-CIRBPI转染细胞48 h,然后加入激活雷帕霉素靶蛋白复合物1(mTORC1)活性的胰岛素。通过RT-qPCR检测CIRBP的mRNA表达,Western blot检测CIRBP、磷酸化核糖体蛋白S6(p-S6Ser235/236)和磷酸化4E结合蛋白1(p-4EBP1Thr37/46)蛋白水平变化,Ki67免疫荧光染色和CCK-8实验检测细胞增殖,划痕实验检测细胞迁移,HE染色检测颈动脉内膜增生程度。结果:沉默CIRBP后,VSMCs的活力下降,Ki67阳性细胞比率降低,细胞迁移速度减慢,同时mTORC1活性下降。加入胰岛素恢复mTORC1活性后,细胞活力、Ki67阳性细胞率和细胞迁移速度下降幅度减弱。损伤小鼠颈总动脉内皮后CIRBP表达增加,体内干扰小鼠CIRBP表达后,小鼠血管内皮损伤后内膜增生程度减轻。结论:CIRBP通过mTORC1途径加强小鼠VSMCs增殖及迁移,促进小鼠血管损伤后血管内膜增生。

AIM:To investigate the regulatory effect of cold-inducible RNA binding protein (CIRBP) on the proliferation and migration of mouse vascular smooth muscle cells (VSMCs),and to explore its mechanism.METHODS:Male C57BL/6J mice were divided into 4 groups:sham+adenovirus-negative control (AD-NC) group,sham+adenovirusCIRBP inhibitor (AD-CIRBPI) group,carotid vascular injury+AD-NC group,and carotid vascular injury+AD-CIRBPI group,with 5 mice in each group.After the injury model was established,the mice were transfected with AD-NC or AD-CIRBPI.The CIRBP expression and vascular intimal hyperplasia were measured at 28 d after injury.Mouse VSMCs were divided into AD-NC and AD-CIRBPI group.The cells of these two groups were transfected with AD-NC and AD-CIRBPI for 48 h,respectively.The activity of mammalian target of rapamycin complex 1 (mTORC1) was restored by insulin.The mRNA expression of CIRBP was explored by RT-qPCR.The levels of CIRBP,phosphorylated ribosomal protein S6 (pS6Ser235/236) and phosphorylated 4E-binding protein 1 (p-4EBP1Thr37/46) were detected by Western blot.The cell proliferation was explored by Ki67 immunofluorescence staining and CCK-8 assay.The cell migration was determined by scratch test.The carotid intimal hyperplasia was determined by HE staining.RESULTS:The viability of VSMCs,the ratio of Ki67-positive cells,wound-healing rate and the activity of mTORC1 were decreased after silencing of CIRBP.However,the in?hibitory effects of CIRBP silencing on cell viability,ratio of Ki67-positive cells and wound-healing rate were attenuated af?ter restoring the activity of mTORC1 by insulin.The expression of CIRBP was increased after mouse common carotid vas?cular injury.The vascular intimal hyperplasia was attenuated after interfering with the expression of CIRBP in vivo.CON?CLUSION:CIRBP promotes the proliferation and migration of mouse VSMCs,and vascular intimal hyperplasia after mouse common carotid vascular injury through the mTORC1 pathway.