miR⁃204通过调控ZEB1抑制胃癌血管生成的机制研究

Mechanism of miR⁃204 inhibiting angiogenesis of gastric cancer by regulating ZEB1

目的探讨miR⁃204通过调控E盒结合锌指蛋白1(ZEB1)抑制胃癌血管生成的机制,为胃癌的治疗提供参考依据。方法采用TCGA数据库和免疫组化染色分析临床胃癌患者癌组织和癌旁组织样本中miR⁃204和ZEB1的表达水平;UALCAN数据库分析不同临床病理分期和淋巴转移胃癌患者中miR⁃204和ZEB1的表达水平,以及二者与患者总生存期(OS)的相关性。血管生成实验检测人脐静脉内皮细胞(HUVEC)经miR⁃204过表达和敲减HGC⁃27细胞的条件培养基培养后血管生成能力的变化;Western blot检测miR⁃204对HGC⁃27细胞中ZEB1、VEGFR1和VEGFR2蛋白表达的影响。结果TCGA数据库和免疫组化染色结果显示,与癌旁组织相比,胃癌组织中miR⁃204表达明显降低,而ZEB1表达明显上调,差异均有统计学意义(t=16.398、14.284,P均<0.001);经Pearson相关性分析结果显示,二者表达成负相关(r=-0.827,P<0.001)。UALCAN数据库分析结果显示,miR⁃204在不同临床病理分期和淋巴转移胃癌患者中的表达较正常组织均显著降低(F=17.184、18.633,P均<0.001),且与胃癌患者的OS成正相关(r=0.547,P<0.05);而ZEB1在不同临床病理分期和淋巴转移胃癌患者中的表达较正常组织均明显升高(F=17.817、18.538,P均<0.001),且与胃癌患者的OS成负相关(r=-0.728,P<0.001)。此外,qRT⁃PCR和体外共培养实验结果显示,HUVEC细胞采用miR⁃204过表达条件培养基培养后的血管生成能力明显降低(P<0.001),而采用miR⁃204敲减条件培养基培养后的血管生成能力显著上调(P<0.01)。Western blot检测结果显示,miR⁃204高表达可显著抑制HGC⁃27细胞中ZEB1的表达,并诱导VEGF/VEGFR信号通路失活(P<0.01)。结论miR⁃204表达上调可通过诱导ZEB1/VEGF/VEGFR信号通路失活抑制胃癌血管生成。

Objective To investigate the underlying mechanism of miR⁃204 inhibiting angiogenesis of gastric cancer by regulating zinc finger E⁃box⁃binding protein 1(ZEB1).Methods The expression levels of miR⁃204 and ZEB1 in tumors and adjacent tissues of clinical gastric cancer patients were analyzed by using TCGA database and immunohistochemical staining.The expression levels of miR⁃204 and ZEB1 in gastric cancer patients with different clinicopathological stages and lymphatic metastasis,and their correlation with overall survival(OS)were analyzed by using the UALCAN database.Angiogenesis assay was used to detect the changes of angiogenesis ability of human umbilical vein endothelial cells(HUVEC)after cultured in conditioned medium with miR⁃204 overexpression and knockdown in HGC⁃27 cells.The effects of miR⁃204 on the protein expression levels of ZEB1,VEGFR1 and VEGFR2 in HGC⁃27 cells were detected by western blot.Results TCGA database and immunohistochemical staining results showed that compared with adjacent tissues,the expression levels of miR⁃204 in gastric cancer tissues was significantly decreased(P<0.001),while the expression levels of ZEB1 was significantly upregulated(P<0.001),and Pearson correlation analysis result showed a negative correlation between miR⁃204 and ZEB1 expression levels(r=-0.827,P<0.001).The results of UALCAN database analysis showed that the expression levels of miR⁃204 in gastric cancer patients with different clinicopathological stages and lymphatic metastasis were significantly decreased than that in normal tissues(F=17.817,18.538;all P<0.001),and was positively correlated with OS in gastric cancer patients(r=0.547,P<0.05),while the expression levels of ZEB1 in gastric cancer patients with different clinicopathological stages and lymphatic metastasis were significantly increased and higher than that in normal tissues(F=17.817,18.538;all P<0.001),and was negatively correlated with OS in gastric cancer patients(r=-0.728,P<0.001).In addition,qRT⁃PCR and in vitro co⁃culture results showed that the angiogenesis capacity of HUVEC cells cultured with miR⁃204 overexpression conditioned medium was significantly decreased(P<0.001),while the angiogenesis capacity of HUVEC cells cultured with miR⁃204 knockdown conditioned medium was significantly increased(P<0.01).Finally,western blot results showed that miR⁃204 overexpression significantly inhibited ZEB1 expression and induced the inactivation of VEGF/VEGFR signaling pathway in HGC⁃27 cells(P<0.001).Conclusion Upregulation of miR⁃204 expression could inhibit angiogenesis of gastric cancer by inducing the inactivation of ZEB1/VEGF/VEGFR signaling pathway.