miR-24对人脐静脉内皮细胞增殖、转移及自噬的影响

Effects of miR-24 on cell proliferation,metastasis and autophagy of HUVECs

目的探讨miR-24对人脐静脉内皮细胞(HUVECs)增殖、转移、自噬的影响。方法将HUVECs随机分为空白对照组、雷帕霉素+miR-24高表达组、雷帕霉素组。空白对照组不给予任何处理;雷帕霉素组的HUVECs用1 000 nmol/L雷帕霉素处理6 h建立自噬模型。雷帕霉素+miR-24高表达组的HUVECs先转染miR-24高表达质粒,待转染成功后以同样方法建立自噬模型;用CCK-8法检测HUVECs增殖能力,细胞划痕试验检验细胞的转移能力,进一步用免疫组织化学和Western blotting法检测HUVECs自噬蛋白微管相关蛋白1轻链3(LC3)和Beclin-1的蛋白表达水平,采用透射电镜观察细胞内部自噬小体。结果与空白对照组相比,雷帕霉素组、雷帕霉素+miR-24高表达组OD值、细胞迁移比例降低、Beclin-1蛋白及LC3Ⅱ蛋白表达量上升(P<0.01或<0.05)。与雷帕霉素组相比,雷帕霉素+miR-24高表达组OD值、细胞迁移比例下降,Beclin-1、LC3Ⅱ蛋白表达量下降(P<0.01或<0.05)。透射电镜观察结果显示,空白对照组和雷帕霉素+miR-24高表达组的细胞质内各细胞器分布基本正常,细胞核形态也正常,细胞质中未见明显自噬小体;雷帕霉素组细胞质中可见明显的空泡状结构、包含部分双层膜结构的自噬小体。结论 miR-24可显著抑制HUVECs的增殖、转移及自噬。

To investigate the effects of miR-24 on the cell proliferation, metastasis and autophagy of human umbilical vein endothelial cells （HUVECs）.Methods HUVECs were randomly divided into the blank control group, rapamycin ＋ miR-24 high expression group and rapamycin group.The control group was not treated.HUVECs in the rapamycin group were treated with 1000 nmol/L rapamycin for 6 h to establish an autophagic model.HUVECs in the rapamycin ＋ miR-24 high expression group were transfected with miR-24 high expression plasmid.After successful transfection, the cells were treated with 1 000 nmol/L rapamycin for 6 h to establish the autophagic models.The proliferation ability of HUVECs was detected by CCK-8, and the cell migration ability was measured by cell scratch test, the protein expression levels of microtubule-associated protein 1 light chain 3 （LC3） and Beclin-1 in HUVECs were detected by immunohistochemistry and Western blotting.The autophagic bodies were observed by transmission electron microscopy.Results Compared with the blank control group, the OD value and migration ratio of the rapamycin group and the rapamycin ＋ miR-24 high expression group were decreased and the expression of Beclin-1 protein and LC3 II protein was increased （P〈0.01 or P〈0.05）.Compared with the rapamycin group, the OD value and migration ratio of the rapamycin ＋ miR-24 high expression group were decreased, and the expression of Beclin-1 and LC3 II protein was decreased （P〈0.01 or P〈0.05）.The results of transmission electron microscopy showed that the distribution of each organelle in the cytoplasm of the blank control group and the rapamycin ＋ miR-24 high expression group was normal, the nucleus morphology was normal and the autophagic bodies were not found in the cytoplasm.In the the rapamycin group, the obvious vacuolar structure and the autophagosome containing part of the bilayer structure conld be seen in the cytoplasm.Conclusion miR-24 significantly inhibits the proliferation, metastasis and autophagy of HUVECs.