shRNA抑制p115表达对胃癌体外血管形成的影响

Influence of shRNA-mediated p115 gene silencing on angiogenesis in gastric cancer cells in vitro

目的初步探讨胃癌BGC-823细胞高尔基体囊泡转运蛋白(golgi-vesicular transport protein,p115)对胃癌血管形成的影响及其可能机制。方法采用脂质体介导法将p115 shRNA-1318转染入胃癌BGC-823细胞株,经G418筛选出稳定转染的细胞株。利用Western blot、RT-PCR和细胞免疫荧光方法检测转染后的p115抑制效应及其对MIF、p-Akt、VEGF-A的调控情况;采用细胞活性检测实验、Transwell体外侵袭实验和血管形成实验方法分别检测转染p115 shRNA的胃癌BGC-823细胞对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVECs)增殖,迁移和血管形成的影响。结果 p115 shRNA-1318转染后胃癌BGC-823细胞p115、MIF、p-Akt和VEGF-A的蛋白及mRNA表达明显抑制(P<0.01);细胞免疫荧光:与对照组相比,p115 shRNA组p115、MIF及VEGF-A的色泽暗红且抑制率分别为63%、65%、68%;细胞活性检测:与对照组相比,p115 shRNA组HUVECs增殖能力明显降低(P<0.05);Transwell侵袭实验:p115 shRNA组HUVECs较空白对照组,阴性对照组(shNC)细胞侵袭能力明显降低,24 h穿过Transwell的细胞数分别为:(39±5)、(174±4)、(179±4),与对照组相比差别具有统计学意义(P<0.05);血管形成实验:p115 shRNA组HUVECs不能在Matrigel胶上形成网状结构,空白对照组、shNC组成管指数分别为:(1 289±37)、(1 329±33),p115 shRNA组成管指数为:(422±41),与对照组相比差别具有统计学意义(P<0.05)。结论 p115基因沉默下调胃癌的VEGF-A表达及抑制血管生成;其分子机制可能与MIF通过PI3K/Akt信号通路途径调节胃癌血管形成有关。

Objective To investigate the influence of Golgi-vesicular transport protein（p115） in gastric cancer cell line BGC-823 on the angiogenesis of gastric cancer and its possible mechanism.Methods BGC-823 cells were divided into a blank control group,a negative control group,and a p115 shRNA-1318-transfected group.In the p115 shRNA-transfected group,p115 shRNA-1318 was transferred into BGC-823 cells by liposome-mediated transfection.The well-transfected BGC-823 cells were screened out using G418.Western blotting was employed to analyze the protein expression levels of p115,MIF,p-Akt,and VEGF-A in BGC-823 cells.RT-PCR was employed to analyze the mRNA expression levels of p115,MIF and VEGF-A in BGC-823 cells.Cell immunofluorescence assay was employed to analyze the distributions and inhibitory rates of p115,MIF and VEGF-A in BGC-823 cells.The effect of p115 shRNA-1318-transfected BGC-823 cells on the proliferation,migration and angiogenesis of human umbilical vein endothelial cells（HUVECs） were analyzed by cellular activity assay,Transwell in vitro invasion assay,and Matrigel assay,respectively.Results The protein expression levels of p115,MIF,p-Akt,and VEGF-A as well as the mRNA expression levels of p115,MIF,and VEGF-A were significantly inhibited in the p115 shRNA-transfected group（P0.01）.As proven by cell immunofluorescence assay,the expression levels of p115,MIF,and VEGF-A in the p115 shRNA-transfected group were significantly inhibited when compared with those in the control groups,with inhibitory rates of 63%,65%,and 68%,respectively.The proliferation rate of HUVECs in the p115 shRNA-transfected group was significantly lower than those in the control groups（P0.05）.As compared with those in the control groups,the BGC-823 cells in the p115 shRNA-transfected group had significantly decreased invasion ability（P0.05）.The numbers of the cells passing through Transwell chamber within 24 h in the p115 shRNA-transfected group,blank control group,and negative control group were 39±5,174±4,and 179±4,respectively.The capillary-like structures formed in the p115 shRNA-transfected group were significantly fewer than those formed in the blank control group and negative control group（422±41 vs 1 289±37 and 1 329±33,P0.05）.Conclusion Gene silencing of p115 can down-regulate the expression of MIF and VEGF-A in gastric cancer cells and suppress the angiogenesis of gastric cancer.The molecular mechanism is probably related to the fact that MIF adjusts the angiogenesis of gastric cancer through PI3K/Akt signaling pathway.