肿瘤相关性成纤维细胞高表达LncRNA ZEB1-AS1促进肺腺癌血管生成的功能机制

Function and mechanism of cancer associated fibroblasts highly expressing LncRNA ZEB1-AS1 to promote angiogenesis in lung adenocarcinoma

目的当前关于ZEB1-AS1对肺腺癌血管生成的作用机制尚未明确。文中研究肿瘤相关性成纤维细胞中长链非编码RNALncRNA ZEB1-AS1的表达及其对肺腺癌细胞和血管内皮细胞功能的影响。方法采取RT-PCR检测ZEB1-AS1在肺腺癌组织与配对癌旁正常组织以及肺腺癌细胞系,肿瘤相关性成纤维细胞(CAFs)以及配对的正常成纤维细胞中的表达情况。利用特异性小干扰RNA(siRNA)构建ZEB1-AS1低表达CAF细胞系。通过血管生成实验和A549细胞侵袭迁移实验研究CAFs中的ZEB1-AS1对HUVEC血管形成的影响和对肺腺癌浸润的影响。通过酶联免疫吸附反应实验(ELISA)测定沉默ZEB1-AS1CAFs中可能的血管相关性细胞因子,并通过双荧光素酶报告基因实验探究ZEB1-AS1在血管生成调节方面的机制。结果LncRNAZEB1-AS1在肿瘤组织和CAFs中高表达(24.89±19.11),在正常组织和肺腺癌细胞系中低表达(7.09±6.95)。在CAFs中沉默ZEB1-AS1能够抑制HUVEC的血管形成,进而抑制肺腺癌细胞A549的侵袭迁移;并且沉默ZEB1-AS1 CAFs上清中血管内皮生长因子A(VEGFA)分泌减少。双荧光素酶报告基因实验以及统计学分析显示,ZEB1-AS1可能通过竞争性结合miR-505-3p从而促进VEGFA的分泌。结论LncRNA ZEB1-AS1是CAFs高表达的肿瘤血管形成相关的lncRNA,抑制ZEB1-AS1能够抑制HUVEC的血管形成和A549的侵袭转移。ZEB1-AS1可能是肺腺癌潜在的预后标志和治疗靶点。

Objective To study the expression of long non-coding RNA(lncRNA)LncRNA ZEB1-AS1 in cancer associated fibroblasts(CAFs)and its effect on the function of lung adenocarcinoma(LUAD)cells and vascular endothelial cells.Methods The expression levels of ZEB1-AS1 in LUAD tissues and primary fibroblasts were determined by quantitative reverse transcription polymerase chain reaction(RT-PCR)and in situ hybridization(ISH).Small interfering RNA(siRNA)was used to silence ZEB1-AS1 in CAFs.The effect of ZEB1-AS1 on HUVEC angiogenesis and on lung adenocarcinoma infiltration was investigated by angiogenesis assay and A549 invasion and migration assay.Enzyme linked immunosorbent assay(ELISA)and dual-luciferase reporter gene assay were used to investigate the mechanisms of ZEB1-AS1 in regulating angiogenesis.Results LncRNA ZEB1-AS1 was highly expressed in LUAD tissues and CAFs compared to adjacent normal tissues and LUAD cell lines.Knockdown of ZEB1-AS1 in CAFs inhibited the angiogenesis of HUVEC,the invasion and migration ability of A549,and inhibited secretion of vascular endothelial growth factor A(VEGFA)in CAFs.Dual luciferase reporter assays showed that ZEB1-AS1 promoted VEGFA secretion by competitively binding miR-505-3p.Conclusion LncRNA ZEB1-AS1 is a tumor angiogenesis-associated lncRNA highly expressed in CAFs.Knock down of ZEB1-AS1 suppresses the migration and invasion ability of A549 cells via inhibiting the angiogenesis of HUVEC.ZEB1-AS1 may be a potential prognostic marker and therapeutic target for LUAD.