miR-181a调控sirt1对缺氧条件下大鼠脑微血管内皮细胞增殖和凋亡的影响

Effects of miR-181a regulating SIRT1 on the proliferation and apoptosis of rat brain microvascular endothelial cells under hypoxia

目的探究miR-181a对缺氧条件下大鼠脑微血管内皮细胞(rBMECs)增殖、凋亡的影响及可能的作用机制。方法取对数生长期的rBMECs分为对照组、模型组、NC inhibitor组、miR-181a inhibitor组、miR-181a inhibitor+si-NC组、miR-181a inhibitor+si-SIRT1组。体外模拟脑缺氧微环境,qRT-PCR法检测6组细胞中miR-181a和沉默信息调节因子1(SIRT1)mRNA的表达;MTT法、流式细胞术分别检测6组细胞增殖、凋亡情况;荧光分光光度计检测6组细胞荧光强度,并通过计算透过Transwell小室的异硫氰酸荧光素标记的葡聚糖(FITC-Dextran)的浓度评价rBMECs的屏障功能;DCFH-DA荧光探针检测细胞内活性氧(ROS)水平;生化法检测超氧化物歧化酶(SOD)活性、丙二醛(MDA)和还原型谷胱甘肽(GSH)水平;Western blot检测血管内皮钙粘蛋白(VE-cadherin)、SIRT1和凋亡相关蛋白的表达。结果与对照组比较,模型组rBMECs中miR-181a的表达、细胞凋亡率、FITC-Dextran浓度、ROS、MDA水平、Bcl-2相关X蛋白(Bax)、半胱天冬氨酸蛋白酶3(Caspase-3)表达显著升高(P<0.05),SIRT1 mRNA和蛋白表达、细胞增殖率、SOD、GSH水平、VE-cadherin、B细胞淋巴瘤-2(Bcl-2)表达显著降低(P<0.05);与模型组比较,miR-181a inhibitor组细胞中miR-181a的表达、细胞凋亡率、FITC-Dextran浓度、ROS、MDA水平、Bax、Caspase-3表达明显降低(P<0.05),SIRT1 mRNA和蛋白表达、细胞增殖率、SOD、GSH水平、VE-cadherin、Bcl-2表达明显升高(P<0.05);使用si-SIRT1干扰SIRT1的表达能明显逆转miR-181a inhibitor对缺氧诱导的rBMECs增殖、凋亡及屏障功能的作用。结论下调miR-181a的表达可促进缺氧诱导的rBMECs增殖、抑制rBMECs凋亡;其作用机制可能与上调SIRT1的表达有关。

Objective To investigate the effects of miR-181a regulating SIRT1 on the proliferation and apoptosis of rat brain microvascular endothelial cells(rBMECs)under hypoxia and its possible action mechanism.Methods The rBMECs at logarithmic growth phase were divided into control group,model group,NC inhibitor group,miR-181a inhibitor group,miR-181a inhibitor+si-NC group,miR-181a inhibitor+si-SIRT1 group.In vitro simulated brain hypoxia microenvironment,qRT-PCR method was used to detect the expression of miR-181a and silence information regulator 1(SIRT1)mRNA;MTT method and flow cytometry were used to detect cell proliferation and apoptosis;fluorescence spectrophotometer was used to detect the fluorescence intensity,and the barrier function of rBMECs was evaluated by calculating the concentration of fluorescein isothiocyanate-labeled dextran(FITC-Dextran)passing through Transwell chamber;DCFH-DA fluorescent probe was used to detect the levels of reactive oxygen species(ROS);biochemical method was used to detect superoxide dismutase(SOD)activity,and the levels off malondialdehyde(MDA)and reduced glutathione(GSH);Western Blot was used to detect the expression levels of VE-cadherin,SIRT1 and apoptosis-related proteins.Results Compared with those in control group,the expression levels of miR-181a,apoptosis rate,FITC-Dextran concentration,ROS and MDA levels,Bcl-2 associated X protein(Bax),and Caspase-3 expression in model group were increased significantly(P<0.05),however,the expression levels of SIRT1 mRNA and protein,cell proliferation rate,SOD and GSH levels,VE-cadherin,and B-cell lymphoma-2(Bcl-2)expression were decreased significantly(P<0.05).Compared with those in model group,the expression levels of miR-181a,apoptosis rate,FITC-Dextran concentration,ROS and MDA levels,Bax,and Caspase-3 expression in miR-181a inhibitor group were decreased significantly(P<0.05),while,the expression levels of SIRT1 mRNA and protein,cell proliferation rate,SOD and GSH levels,VE-cadherin,and Bcl-2 expression were increased significantly(P<0.05).Using si-SIRT1 to interfere with the expression of SIRT1 could significantly reverse the effects of miR-181a inhibitor on the proliferation,apoptosis and barrier function of rBMECs induced by hypoxia.Conclusion Down-regulating the expression of miR-181a can promote the proliferation of rBMECs induced by hypoxia and inhibit the apoptosis of rBMECs,and its action mechanism may be related to the up-regulation of the expression of SIRT1.