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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 effect of golgi-vesicular transport protein (p115) on gastric carcinogenesis and its possible mechanism in gastric cancer BGC-823 cells. Methods p115 shRNA-1318 was transfected into gastric cancer cell line BGC-823 by liposome-mediated method. Stably transfected cell lines were selected by G418. The inhibitory effect of p115 and its regulation on the expression of MIF, p-Akt, VEGF-A were detected by Western blot, RT-PCR and immunofluorescence staining. The cell viability assay, Transwell invasion assay and angiogenesis assay Methods The effects of p115 shRNA transfected BGC-823 cells on the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were detected. Results The protein and mRNA expression of p115, MIF, p-Akt and VEGF-A in gastric cancer BGC-823 cells transfected with p115 shRNA-1318 were significantly inhibited (P <0.01). Immunofluorescence: Compared with the control group, p115 shRNA group p115, MIF and VEGF-A were 63%, 65%, 68%, respectively. Cell viability was detected in HUVECs transfected with p115 shRNA (P <0.05) Invasion experiment: The invasion ability of HUVECs in p115 shRNA group was significantly lower than that in blank control group and negative control group (shNC). The numbers of cells passing through Transwell in the p115 shRNA group were (39 ± 5), (174 ± 4), (179 ± 4), compared with the control group, the difference was statistically significant (P <0.05). Angiogenesis assay: HUVECs in p115 shRNA group could not form reticular structures on Matrigel, and the index of tube in blank control group and shNC group was (1 289 ± 37), (1 329 ± 33) and p115 shRNA (422 ± 41), respectively. The difference was statistically significant (P <0.05) compared with the control group. Conclusion The silencing of p115 down-regulates the expression of VEGF-A and the angiogenesis in gastric cancer. The molecular mechanism may be related to the regulation of vascular endothelial growth by MIF through the PI3K / Akt signaling pathway.