ox-Lp(a)通过上调miR-125a-5p靶向抑制10,11-转位酶2增加单层血管内皮细胞通透性

Ox-Lp(a) increases permeability of monolayer vascular endothelial cells by upregulating miR-125a-5p expression and targeted inhibiting 10,11-translocation enzyme 2

目的探讨ox-Lp(a)损伤血管内皮细胞的表观遗传调控机制。方法生物信息学分析和筛选与10,11-转位酶2(TET2)mRNA 3’-UTR靶向结合的候选miRNA,荧光素酶报告基因系统验证其结合的靶向性;以0 mg/L、25 mg/L、50 mg/L、100 mg/L及200 mg/L的ox-Lp(a)与HUVEC-12内皮细胞孵育24 h,或用100 mg/L ox-Lp(a)与HUVEC-12内皮细胞孵育0 h、6 h、12 h、24 h及48 h,qRT-PCR和Western blot分别检测TET2 mRNA和蛋白的表达水平。qRT-PCR检测hsa-miR-125a-5p表达水平,以5hmc水平分析TET2活性的变化,Transwell检测ox-Lp(a)对单层血管内皮细胞通透性的影响。结果生物信息学分析和荧光素酶报告基因验证结果表明TET2为hsa-miR-125a-5p的靶基因,且hsa-miR-125a-5p与TET2 mRNA的3’-UTR结合的自由能值低(-30.1 kcal/mol)。ox-Lp(a)呈剂量和时间依赖性抑制TET2蛋白和mRNA的表达水平,以100 mg/L ox-Lp(a)作用HUVEC-12内皮细胞24 h的效果最佳;100 mg/L ox-Lp(a)作用HUVEC-12内皮细胞24 h后,TET2活性显著下降,且显著上调hsamiR-125a-5p的表达。anti-hsa-miR-125a-5p能逆转ox-Lp(a)对HUVEC-12内皮细胞TET2蛋白和mRNA表达水平的抑制作用和活性下降。ox-Lp(a)显著增加单层血管内皮细胞通透性,但可被anti-hsa-miR-125a-5p部分逆转。结论ox-Lp(a)通过上调hsa-miR-125a-5p并与TET2 mRNA 3’-UTR靶向性结合,抑制TET2蛋白和mRNA的表达水平及活性,从而增加单层血管内皮细胞通透性。

Aim To investigate the epigenetic regulation mechanism of oxidized lipoprotein（a） [ox-Lp（a） ]injury on vascular endothelial cells. Methods Bioinformatics and luciferase reporter gene were used to screen candidate microRNA binding to 10,11-translocation enzyme 2（TET2） mRNA 3＇-UTR and verify their targeted binding tendency. 0 mg/L,25 mg/L,50 mg/L and 100 mg/L of ox-Lp（a） were incubated with HUVEC-12 vascular endothelial cell line for 24 h,or incubated with HUVEC-12 vascular endothelial cell line with 100 mg/L ox-Lp（a） for 0 h,6 h,12 h,24 h,48 h respectively. qRT-PCR and Western blot were used to detect TET2 mRNA and protein expression levels. The expression of hsa-miR-125 a-5 p was detected by qRT-PCR. The change of TET2 activity was analyzed by detecting 5 hmc level. Transwell was used to detect the permeability of monoclonal vascular endothelial cells. Results Bioinformatic analysis and luciferase reporter assay showed that TET2 was the target gene of hsa-miR-125 a-5 p,and the binding energy of hsa-miR-125 a-5 p to the 3＇-UTR of TET2 mRNA was low（-30.1 kcal/mol）. The activity of TET2 protein and mRNA was inhibited by ox-Lp（a） in the dose and time-dependent manner. The best reaction dose and time of ox-Lp（a） was100 mg/L and 24 h. The activity of TET2 was down-regulated by 100 mg/L ox-Lp（a）,while the expression of hsa-miR-125 a-5 p was significantly up-regulated,and anti-hsa-miR-125 a-5 p could reverse it. Ox-Lp（a） significantly increased the permeability of monolayer endothelial cells,but could be partially reversed by anti-hsa-miR-125 a-5 p. Conclusion Ox-Lp（a） inhibited the expression and activity of TET2 and increased the permeability of monolayer vascular endothelial cells by up-regulating hsa-miR-125 a-5 p expression which targeted binding to 3＇-UTR of TET2 mRNA.