E2F调控lncRNA HOTAIR在高磷诱导VSMCs钙化中的作用机制研究  

Role and mechanism of E2F in high phosphorus-induced calcification of VSMCs by targeting lncRNA HOTAIR

作　　者：陈艳霞[1] 柯本 涂卫平[1] 陈艳[1] 黄翀[1] 朱淑英[1] CHEN Yanxia;KE Ben;TU Weiping;CHEN Yan;HUANG Chong;ZHU Shuying(Department of Nephrology,the Second Affiliated Hospital of Nanchang University,Nanchang 330006,China)

机构地区：[1]南昌大学第二附属医院肾脏内科,江西南昌330006

出　　处：《中国病理生理杂志》2023年第1期96-102,共7页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.82260152);江西省管理工程技术研究中心(No.20164BCD40095)。

摘　　要：目的:探讨转录因子E2F调控长链非编码RNA(long noncoding RNA, lncRNA) HOTAIR在高磷(high phosphorus, HP)诱导血管平滑肌细胞(vascular smooth muscle cells, VSMCs)钙化中的作用及其机制。方法:体外培养人VSMCs,采用HP(10 mmol/L β-磷酸甘油酯)诱导构建VSMCs钙化模型,设置实验分组:正常对照(control)组和HP组。采用试剂盒进行钙沉积含量测定;RT-qPCR检测VSMCs中E2F mRNA、lncRNA HOTAIR和Klotho mRNA的表达;Western blot检测E2F和Klotho蛋白的表达。过表达E2F,设置实验分组:control组、HP组、HP+空载(HP+Adlaz)组和HP+过表达E2F(HP+AdE2F)组。茜素红染色评估细胞钙化并进行钙沉积含量测定;RT-qPCR检测VSMCs中E2F mRNA、lncRNA HOTAIR和Klotho mRNA的表达;Western blot检测E2F和Klotho蛋白的表达;双萤光素酶报告基因检测验证E2F、lncRNA HOTAIR和Klotho的靶向关系。结果:与control组相比较,HP组细胞呈现明显的钙化,钙沉积含量增加;RT-qPCR显示E2F mRNA、lncRNA HOTAIR和Klotho mRNA在HP组中表达下调;Western blot提示E2F和Klotho蛋白在HP组表达下调。过表达E2F后,HP+AdE2F组细胞较HP+Adlaz组减轻,钙沉积含量减少;RT-qPCR提示lncRNA HOTAIR和Klotho mRNA在HP+AdE2F组中表达上调,Western blot显示E2F和Klotho蛋白在HP+AdE2F组中表达上调。双萤光素酶报告基因系统检测提示E2F的表达水平与lncRNA HOTAIR呈现显著正相关,同时表明Klotho为lncRNA HOTAIR的靶基因。结论:E2F通过调控lncRNA HOTAIR介导Klotho,从而抑制HP诱导的VSMCs钙化。AIM:To investigate the role and mechanism of E2F in high phosphorus(HP)-induced calcification of vascular smooth muscle cells(VSMCs) by targeting long noncoding RNA(lncRNA) HOTAIR. METHODS:Human VSMCs were cultured in vitro, and VSMC calcification model was induced by HP(10 mmol/L β-glycerol phosphate).The VSMCs were randomly divided into normal control group and HP group. After overexpression of E2F, the VSMCs were divided into 4 groups:normal control group, HP group, HP+Adlaz group, and HP+AdE2F group. The content of calcium deposition was detected by kit. Alizarin red staining was used to evaluate calcification. The RT-qPCR was performed to detect the expression of E2F mRNA, lncRNA HOTAIR and Klotho mRNA. Western blot was used to evaluate the expression of E2F and Klotho proteins. Dual-luciferase reporter assay was used to verify the targeting relationship among E2F, lncRNA HOTAIR and Klotho. RESULTS:Compared with control group, HP group showed a significant increase in calcium deposition. RT-qPCR showed that the expression of E2F mRNA, lncRNA HOTAIR and Klotho mRNA was down-regulated in HP group. Western blot suggested that the expression of E2F and Klotho proteins was down-regulated in HP group. After overexpression of E2F, the calcification and calcium deposition in HP+AdE2F group were less than those in HP+Adlaz group. RT-qPCR suggested that the expression of lncRNA HOTAIR and Klotho mRNA was up-regulated in HP+AdE2F group, and Western blot showed that the expression of E2F and Klotho proteins was up-regulated in HP+AdE2F. Dual-luciferase reporter assay showed that there was a positive correlation between the expression level of E2F and lncRNA HOTAIR. Meanwhile, Klotho is the target gene of lncRNA HOTAIR. CONCLUSION:E2F inhibits the calcification of VSMCs induced by HP via targeting lncRNA HOTAIR to facilitate the expression of Klotho.

关 键 词：E2F转录因子 lncRNA HOTAIR 血管平滑肌细胞 钙化 

分 类 号：R363.2[医药卫生—病理学] R543[医药卫生—基础医学]