microRNA20b调节PPARγ抑制缺氧/复氧诱导的内皮细胞凋亡

摘要

目的:观察microRNA20b对缺氧/复氧诱导内皮细胞凋亡的影响.方法:采用人脐静脉内皮细胞株(HUVECs)建立缺氧/复氧损伤细胞模型.将生长状态良好的HUVECs随机分为正常对照组(Normal组)、缺氧后阴性对照组(HR+NC组)、缺氧后microRNA20b过表达组(HR+20b组)、缺氧后阴性对照抑制组(HR+IN NC组)和缺氧后microRNA20b抑制组(HR+IN 20b组).缺氧前24 h采用lipofectamine2000将Negative Control、microRNA20b mimics、inhibitor Negative Control、microRNA20b inhibitor分别转染入后四组细胞,然后缺氧培养12 h后复氧4 h.采用免疫荧光检测过氧化物酶体增值物受体γ(PPARγ)蛋白的变化,AnnexinV-FITC/PI流式双染法检测细胞的早晚期凋亡率,Western blot检测PPARγ以及凋亡相关蛋白Bax、抗凋亡相关蛋白Bcl-2蛋白表达水平.结果:与Normal组相比,缺氧/复氧损伤后各转染组PPARγ蛋白表达升高,凋亡率升高.与HR+NC组相比,HR+20b组的PPARγ及凋亡相关蛋白Bax表达降低、抗凋亡相关蛋白Bcl-2表达升高,凋亡率降低[(7.70+0.85)%vs(18.20+1.05)%,P<0.05].与HR+IN NC组相比,HR+IN 20b组的PPARγ及凋亡相关蛋白Bax表达升高、抗凋亡相关蛋白Bcl-2表达降低,凋亡率升高[(18.9+3.05)%vs(13.0+2.25)%,P<0.05].结论:microRNA20b靶向作用于PPARγ抑制缺氧/复氧损伤诱导的内皮细胞凋亡.%Objective To investigate the mechanism of microRNA20b reducing apoptosis induced by hypox-ia/reoxygenation injury. Methods The hypoxia/reoxygenation model was created use human umbilical vein en-dothelial cells(HUVECs). HUVECs were divided into 5 groups randomly as normal control group (Normal group), anoxic Negative Control group (HR+NC group), anoxic microRNA20b mimics group (HR+20b group), anoxic in-hibitor Negative Control group (HR+IN NC group) and anoxic microRNA20b inhibitor group (HR+IN 20b group). The last four groups were transfected with Negative control, microRNA20b mimics, inhibitor Negative Control or microRNA20b inhibitor respectively for 24 h. Then the transfected cells were treated with 12 hours hypoxia fol-lowed by 4 hours reoxygenation. Immunofluorescence was used to detect the expression of PPARγ. Annex-inV-FITC/PI double staining apoptosis detection was used to test the apoptosis rate of HUVECs. The expression of Bax、Bcl-2 and PPARγ were determined by western blot. Results The expression of PPARγ was increased after hypoxia/reoxygenation injury compared with Normal group. Compared to HR+NC group , the expression of PPARγ and the apoptosis-related protein Bax were lower in HR+20b group, but the expression of anti-apopto-sis-related protein Bcl-2 was higher. And the apoptosis rate of HR+20b group was lower [(7.70+0.85) % vs (18.20+1.05) %, P<0.05]. Conversely, the cells in HR+IN 20b group expressed higher PPARγ protein and the apoptosis-related protein Bax but lower anti-apoptosis-related protein Bcl-2, also had higher apoptosis rate than HR+IN NC group[(18.9+3.05)% vs (13.0+2.25)%, P<0.05]. Conclusion microRNA20b could prevent the hypoxia/reoxygenation injury-induced apoptosis by regulating the expression of PPARγ.