摘要
目的:探讨LncRNA H19对小鼠主动脉平滑肌细胞(MOVAS)钙化过程中表型转化的调控机制。方法:培养MOVAS细胞系,将细胞分为钙化组与对照组,在钙化组细胞培养基中加入β-甘油磷酸盐(β-GP)14d诱导细胞钙化模型。通过RT-PCR和Western印迹法检测比较两组成骨相关转录因子osterix(OSX)及平滑肌标志物α-SMA的表达水平,检测细胞发生钙化表型转化的程度,通过茜素红染色及碱性磷酸酶(ALP)活性测定检测钙化水平,通过检测wnt1和β-catenin表达水平观察Wnt信号通路的变化。后续通过si-RNA技术对H19表达进行敲减,将细胞分为siNC组、siNC+β-GP组、siH19+β-GP组,通过上述方法比较三组中OSX、α-SMA、wnt1和β-catenin表达差异及钙化情况,进一步研究H19与动脉钙化的相关性及发生机制。结果:与对照组相比,钙化组LncRNA H19表达量明显升高,OSX表达增加,α-SMA表达下调,同时Wnt信号通路的关键蛋白wnt1和β-catenin表达增高(P<0.05)。敲减LncRNA H19后,OSX表达水平较普通钙化组降低,α-SMA表达有所回升,钙化程度减低,Wnt信号通路关键蛋白表达降低(P<0.05)。结论:LncRNA H19可促进MOVAS的钙化表型转化及动脉钙化的发生,其机制可能与激活Wnt/β-catenin/OSX轴有关。
Objective:To investigate the regulatory mechanism of LncRNA H19 on phenotypic transformation in mouse aortic smooth muscle cell(MOVAS)calcification.Methods:The MOVAS cell lines were cultured and divided into calcified group and control group.The calcified cell model was induced by addingβ-glycerophosphate(β-GP)into the calcified cell medium for 14 days.The expression levels of osterix(OSX)andα-SMA,the smooth muscle marker,were detected by RT-PCR and Western blot to detect the degree of calcification phenotypic transformation.The calcification level was detected by alizarine red staining and alkaline phosphatase(ALP)activity.The changes of Wnt signaling pathway were observed by detecting the expression levels of WNT1 andβ-catenin.Subsequently,the expression of H19 was knocked down by si-RNA technique,and the cells were divided into siNC group,siNC+β-GP group and siH19+β-GP group.The expression differences of OSX,α-SMA,WNT1,β-catenin and calcification in the three groups were compared by the above methods.Results:Compared with the control group,the expression of LncRNA H19,OSX,WNT1 andβ-catenin were significantly increased in calcified MOVAS group,whileα-SMA decreased(P<0.05).After knocking down the expression of LncRNA H19 by gene editing technology,the expression level of OSX decreased,α-SMA increased and the degree of calcification was weakened(P<0.05).At the same time the expression of key proteins in WNT signaling pathway decreased(P<0.05).Conclusions:LncRNA H19 promotes calcification phenotype transformation and arterial calcification of MOVAS and its potential mechanism may be related to activation of Wnt1/β-catenin/OSX axis.
作者
乔家明
管强麟
吴思婧
马茜
杨佳奇
黄鑫
周玉杰
QIAO Jiaming;GUAN Qianglin;WU Sijing;MA Qian;YANG Jiaqi;HUANG Xin;ZHOU Yujie(Department of Cardiology,Beijing Anzhen Hospital,Capital Medical University,2Beijing Institute of Heart Lung and Blood Vessel Diseases,Beijing 100029,China)
出处
《心肺血管病杂志》
CAS
2022年第4期423-429,共7页
Journal of Cardiovascular and Pulmonary Diseases
基金
国家自然科学基金(81770246)
国家自然科学基金(82070293)
北京市自然科学基金(7212027)。
