肺癌细胞外泌体介导miR-17调控血管内皮细胞生物学行为的实验研究

Experimental study on the regulation of proliferation,migration and invasion of vascular endothelial cells by miR-17 mediated by lung cancer exocrine

目的探究肺癌细胞外泌体中miR-17对血管内皮细胞增殖、迁移和侵袭的影响。方法收集15例晚期(Ⅲ~Ⅳ期)肺癌患者,并选择5例健康志愿者作为对照。采集血液样本,分离并鉴定血清外泌体,采用实时定量PCR(qPCR)检测肺癌患者和健康人血清外泌体miR-17的水平。将A549细胞分为2组:对照组(未转染)、外泌体组(利用GV369-miR-17过表达慢病毒,上调A549细胞中的miR-17)。采用Western blotting检测外泌体标志蛋白。将两组分别与人脐静脉内皮细胞(HUVECs)共培养,qPCR检测两组miR-17水平。CCK8法、划痕实验和Transwell小室实验检测内皮细胞活性、划痕愈合率和穿膜细胞数。结果与健康志愿者比较,miR-17在肺癌患者中高表达(4.718±0.245 vs.0.701±0.116),差异有统计学意义(P<0.05)。与对照组比较,外泌体组HUVECs细胞中miR-17表达显著升高(5.969±0.159 vs.1.000±0.146,P<0.05)。CCK8实验结果显示:与对照组比较,外泌体组的活细胞数量在72 h和96 h均显著增加(P<0.05);划痕实验结果显示:24 h外泌体组的迁移率为(28.0±0.57)%,明显高于对照组的(15.0±0.88)%,48 h外泌体组的迁移率为(45.1±1.45)%,明显高于对照组的(32.3±2.03)%,差异具有统计学意义(P<0.01)。Transwell实验结果显示:与对照组比较,miR-17过表达显著增加HUVECs的侵袭能力(202±8 vs.104±2,P<0.01)。结论肺癌细胞外泌体介导的miR-17能够促进血管内皮细胞的增殖、迁移和侵袭,为肺癌的抗血管生成治疗提供潜在分子靶点。

Objective To investigate the effect of miR-17 in lung cancer cell exosomes on the proliferation,migration and invasion of vascular endothelial cells.Methods Fifteen patients with advanced(stageⅢ-Ⅳ)lung cancer were collected and 5 healthy volunteers were selected as controls.The blood samples was collected,and serum exosomes was isolated and identified.The level of serum exosomes miR-17 in patients with lung cancer and healthy people was detected by real-time quantitative PCR(qPCR).A549 cells were divided into two groups:control group(not transfected)and exosome group(using GV369-miR-17 overexpression lentivirus to up regulate miR-17 in A549 cells).The exosome marker proteins were detected by Western blotting.The two groups were co-cultured with HUVECs,and the level of miR-17 was detected by qPCR.CCK8 method,scratch test and Transwell chamber test were used to detect the activity of endothelial cells,scratch healing rate and the number of transmembrane cells.Results Compared with healthy people,miR-17 was highly expressed in patients with lung cancer(4.718±0.245 vs.0.701±0.116),and the difference was statistically significant(P<0.05).Compared with the control group,the miR-17 expression of HUVECs cells in the exosome group was significantly higher(5.969±0.159 vs.1.000±0.146,P<0.05).The results of CCK8 test showed that compared with the control group,the number of living cells in the exosome group increased significantly at 72 h and 96 h(P<0.05).The results of scratch test showed that the migration rate of 24-hour exosomes group was(28.0±0.57)%,which was significantly higher than that of the control group(15.0±0.88)%,and that of 48 hour exosomes group was(45.1±1.45)%,which was significantly higher than that of the control group(32.3±2.03)%(P<0.01).Transwell experiment showed that compared with the control group,miR-17 overexpression significantly increased the invasive ability of HUVECs(202±8 vs.104±2,P<0.01).Conclusion miR-17 mediated by lung cancer cell exosomes can promote the proliferation,migration and invasion of vascular endothelial cells,and provide a potential molecular target for anti angiogenesis therapy of lung cancer.