微小 RNA34 a 下调沉默信息调节因子 1抑制内皮祖细胞功能的机制研究

摘要

Objective To investigate the mechanism of microRNA34a ( miR-34a) down-regulating silencing of information regulator 1 ( SIRT1) in dysfunction of endothelial progenitor cells during oxidative stress, and provide a new explanation for the dysregulation of miRNA expression during dysfunction of endothelial progenitor cells, so as to find new treatment target for atherosclerosis. Methods In this experiment, endothelial progenitor cells were treated with different concentrations of hydrogen peroxide (H2O2 ), and the survival rate of the cells was detected by CCK-8. The miR-34a mimics, miR-34a inhibitors and their respective control groups were synthesized by chemical synthesis and transfected into endothelial progenitor cells. After the endothelial progenitor cells were stimulated with 500 μM H2 O2 , the expression level of miR-34a was verified by RT-PCR. The apoptosis of miR-34a induced by H2 O2 was detected by flow cytometry. Furthermore, qRT-PCR and Western blotting were used to detect expression levels of mRNA and protein in SIRT1. Results The cell viability decreased with the increase of H2 O2 concentration by CCK-8, which was concentration-dependent. Compared with control groups, the survival rate of cells added with 100μM, 200 μM, 500 μM and 800 μM was respectively (94 ± 2)% , (85 ± 2) % , (78 ± 2)% , (32 ± 1)% . The difference was statistically significant (F=16. 3, P<0. 001). After endothelial progenitor cells were stimulated by H2 O2 , the expression level of miR-34a mimics was higher than that of the control group (t=16, P=0. 003), and the expression level of miR-34a inhibitors was lower than that of the control group ( t =53, P <0. 001 ) . The apoptosis rate of miR-34a mimics group was significantly increased ( t =30. 4, P <0. 001), and the apoptosis rate of miR-34a inhibitors group was significantly decreased (t=8. 7, P<0. 001). The qRT-PCR results showed that the relative expression level of the target gene SIRT 1 in the miR-34a mimics group was significantly lower than that of control group (t=87. 7, P<0. 001), while the miR-34a inhibitors group was significantly higher than that of control group (t=125, P<0. 001). Western Blot results showed that the expression of SIRT 1 protein was significantly decreased in miR-34a mimics group (t=4. 1, P=0. 014), while that was significantly increased in miR-34a inhibitors group( t=7. 1, P=0. 002),compared with control group. Conclusions Under oxidative stress, the miR-34a mimics and inhibitor can modulate the expression of miR-34a, thereby altering the expression of the protein in SIRT1. Endothelial progenitor cell dysfunction may be associated with increased levels of miR-34a and decreased expression of its target protein SIRT1.%目的 分析氧化应激过程中微小RNA34a(miR-34a)下调沉默信息调节因子1( SIRT1)在内皮祖细胞功能障碍中的机制. 方法 本实验采用不同浓度的过氧化氢( H2 O2 )处理内皮祖细胞,通过CCK-8检测细胞的存活率;运用化学合成的方法合成miR-34a模拟物、miR-34a抑制物及其各自对照组转染至内皮祖细胞后,并用500 μM H2 O2 刺激,用RT-PCR验证miR-34a表达水平,利用流式细胞仪检测H2 O2 刺激miR-34a诱导细胞凋亡情况,通过qRT-PCR方法和Western blot技术检测SIRT1中mRNA和蛋白的表达水平. 结果 CCK-8检测发现,细胞存活率随着H2 O2 浓度的增加而逐渐降低,呈现浓度依赖性;与不加H2 O2 相比,加入100、200、500和800 μM的H2 O2 细胞存活率分别为94% ± 2% 、85% ± 2% 、78% ± 2%和32% ± 1% ,差异具有统计学意义(F=16. 3,P<0. 001).H2O2 刺激内皮祖细胞后,转染miR-34a模拟物的表达水平高于其对照组( t=16. 0,P=0. 003),转染miR-34a抑制物的表达水平低于其对照组(t=53. 0,P=0. 000).通过细胞凋亡检测miR-34a模拟物组细胞凋亡率明显升高(t=30. 4,P<0. 001),miR-34a抑制物组细胞凋亡率明显降低(t=8. 7,P<0. 001). qRT-PCR结果显示,miR-34a模拟物组中的靶基因 SIRT1 相对表达量低于其对照组( t =87. 7,P<0. 001),miR-34a抑制物组中的靶基因 SIRT1 相对表达量高于其对照组( t =125. 0, P <0. 001). Western blot结果显示miR-34a的模拟物与其对照组相比,SIRT1蛋白表达量明显下降( t =4. 1,P=0. 014),而 miR-34a 的抑制物与其对照组相比, SIRT1 蛋白表达明显增高( t =7. 1, P =0. 002). 结论 在氧化应激状态下,使用miR-34a的模拟物及抑制物可以调节miR-34a的表达,改变SIRT1中蛋白的表达.内皮祖细胞功能障碍可能与miR-34a水平升高及其靶蛋白SIRT1的表达下降相关.