ACE2通过调节A549细胞中VEGF的表达抑制肺腺癌微血管形成

Regulation of vascular endothelial growth factor expression by angiotensin-converting enzyme 2 inhibited angiogenesis in lung adenocarcinoma microenvironment

目的:研究血管紧张素转换酶2(ACE2)在肺腺癌A549细胞微环境中通过血管内皮生长因子(VEGF)诱导肿瘤血管形成的发生机制。方法:将过表达对照质粒(pcDNA3.1组)、ACE2过表达质粒(pcDNA-ACE2组)、干扰对照质粒(shRNA-NC组)及ACE2干扰质粒(shRNA-ACE2组)转染到肺腺癌A549细胞中,48 h后提取细胞蛋白和上清液,利用蛋白质印迹法和酶联免疫吸附测定分别检测细胞及上清液中VEGF的表达。用上述上清液培养人脐静脉内皮细胞(HUVECs),分为pcDNA3.1组、pcDNA-ACE2组、shRNA-NC组、shRNA-ACE2组及shRNA-ACE2+VEGF antibody组(向shRNA-ACE2组条件培养基加入0.5 mg/L的VEGF中和抗体),分别用MTT、细胞划痕实验、细胞侵袭实验和小管形成实验检测HUVECs的增殖、迁移、侵袭和小管形成能力。结果:pcDNA-ACE2组A549细胞及上清液中VEGF的表达量较pcDNA3.1组减少(P值均<0.05);shRNA-ACE2组A549细胞及上清液中VEGF的表达量较shRNA-NC组增加(P值均<0.05)。pcDNA-ACE2组HUVECs的增殖率、迁移率、侵袭细胞数及小管总长度较pcDNA3.1组下降(P值均<0.05),shRNA-ACE2组HUVECs的增殖率、迁移率、侵袭细胞数及小管总长度较shRNA-NC组上升(P值均<0.05),shRNA-ACE2+VEGF antibody组HUVECs的增殖率、迁移率、侵袭细胞数及小管总长度较shRNA-ACE2组明显降低(P值均<0.05)。结论:ACE2通过下调肺腺癌A549细胞微环境中VEGF的表达,抑制肺腺癌血管内皮细胞的增殖、迁移、侵袭和小管形成能力,抑制肺腺癌体外微血管形成。

Objective To investigate the effect of angiotensin-converting enzyme 2(ACE2)on tumor angiogenesis by regulating vascular endothelial growth factor(VEGF)in the microenvironment of lung adenocarcinoma A549 cells.Methods A549 cells were transfected with pcDNA3.1(control plasmid of overexpression),pcDNA-ACE2(ACE2-overexpressing plasmid),shRNA-NC(control plasmid of interference)or shRNA-ACE2(ACE2-interfering plasm id),the conditioned media were collected after 48 h.The protein expression level of VEGF was quantified by Western blot and the expression level of VEGF in the cell supernatants was quantified by ELISA.Human umbilical vein endothelial cells(HUVECs)were cultured with the conditioned media collected from A549 cells,which were divided into pcDNA3.1 group,pcDNA-ACE2 group,shRNANC group,shRNA-ACE2 group and shRNA-ACE2+VEGF antibody group(add 0.5 mg/L of VEGF neutralizing antibody tothe conditioned media of shRNA-ACE2 group).The proliferative,migratory,invasive,and tube forming abilities of HUVECs were assessed by MTT assay,cell scratch assay,cell invasion assay and tube formation assay,respectively.Results The expression level of VEGF in pcDNA-ACE2 group was decreased compared with that in pcDNA3.1 group(all P<0.05);while the expression level of VEGF in shR NA-ACE2 group was increased compared with that in shRNA-NC group(all P<0.05).In addition,the proliferative rate,migratory rate,invasive cell number and tube total length of HUVECs in pcDNA-ACE2 group were decreased compared with that in pcDNA3.1 group;the proliferative rate,migratory rate,invasive cell number and tube total length of HUVECs in shRNA-ACE 2 group were increased compared with that in shRNA-NC group,and the proliferative rate,migratory rate,invasive cell number and tube total length of HUVECs in shRNA-ACE2+VEGF antibody group were decreased compared with that in shRNA-ACE2 group(all P< 0.05).Conclusions ACE2 is found to inhibit the proliferative,migratory,invasive and tube forming abilities of HUVECs by downregulating the expression of VEGF in the microenvironment of lung adenocarcinoma A 549 cells,thereby inhibiting the microvascular formation of lung adenocarcinoma in vitro.