miR-30a对脂多糖诱导人脐静脉血管内皮细胞自噬的影响

miR-30a attenuates lipopolysaccharide-induced injury of human umbilical vein endothelial cells by inhibiting autophagy

目的探究miR-30a对脂多糖(LPS)诱导人脐静脉血管内皮细胞(HUVEC)自噬的影响及其可能机制。方法以HUVEC为研究对象,分为空白对照组(Control组)、模型组(LPS组)、阴性对照组(转染miR-30a-NC,NC组)和过表达组(转染miR-30a-mimics,mimics组),采用LPS诱导HUVEC建立炎症模型;采用CCK-8法检测细胞存活率;qRT-PCR法检测miR-30a相对表达量;Western blot法检测自噬相关蛋白自噬相关基因(Atg3)、p62、自噬蛋白微管相关蛋白1轻链3(LC3)-II和LC3-I表达水平;激光共聚焦显微镜法观察自噬小体形成变化。结果LPS组细胞存活率明显低于Control组,miR-30a相对表达量明显低于Control组,差异均有统计学意义(均P<0.05)。与Control组比较,LPS组细胞p62蛋白表达水平降低,Atg3蛋白表达水平升高,LC3-II/LC3-I比值升高,差异均有统计学意义(均P<0.05)。转染miR-30a后,与LPS组比较,mimics组细胞存活率明显升高,miR-30a相对表达量明显上升,差异均有统计学意义(均P<0.05)。与LPS组比较,mimics组细胞p62表达水平升高,LC3-II/LC3-I比值和Atg3表达水平降低,差异均有统计学意义(均P<0.05)。与LPS组比较,mimics组细胞胞质内自噬小体显著减少。结论miR-30a能通过抑制自噬减轻LPS诱导HUVEC损伤。

Objective To explore the effects and mechanism of miR-30a on the autophagy in lipopolysaccharide(LPS)-induced injury of human umbilical vein endothelial cells(HUVECs)in vitro.Methods The HUVECs were cultured in vitro and divided into four groups:control group(blank control),LPS group(treated with LPS),NC group(transfected with miR-30a-NC and treated with LPS)and mimics group(transfected with miR-30a-mimics and treated with LPS),The cell injury model was induced by LPS treatment in LPS,NC and mimics groups but not in control group.The proliferation ability of HUVECs were detected by CCK-8 assay,the expression level of miR-30a were detected by qRT-PCR,the protein levels of Atg3,p62,LC3-II and LC3-I were detected by Western blot,the changes of autophagosomes in cells were observed by fluorescence microscopy.Results Compared with control group,the proliferation ability of HUVECs were decreased,the expression of miR-30a and p62 were down regulated,the expression of Atg3 and LC3-Ⅱ/LC3-Ⅰwere up regulated in LPS group(P<0.05).Compared with LPS group,the proliferation ability of HUVECs were increased,the expression of miR-30a and p62 were up regulated,the expression of Atg3 and LC3-Ⅱ/LC3-Ⅰwere down regulated,the numbers of autophagosomes were decreased in mimics group(P<0.05).Conclusion MiR-30a can alleviate LPS induced HUVECs injury by inhibiting autophagy.