骨转移性LUAD细胞通过HGF/YAP信号通路促进血管生成的机制研究  

Mechanism of Bone-metastatic LUAD Cells Promoting Angiogenesis Through HGF/YAP Signaling Pathway

作　　者：邓妍 仇荣 刘星羽 苏英洋 薛阳 杜玉珍 Yan DENG;Rong QIU;Xingyu LIU;Yingyang SU;Yang XUE;Yuzhen DU(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai 201306,China;Department of Laboratory Medicine,Shanghai Jiao Tong University Affiliated Sixth People’s Hospital,Shanghai 201306,China)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]上海交通大学医学院附属第六人民医院医学检验科,上海201306

出　　处：《中国肺癌杂志》2024年第11期805-814,共10页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金项目(No.81974315)资助。

摘　　要：背景与目的肿瘤骨转移早期与骨微环境血管生态位改变密切相关,异常的血管生成加速肿瘤转移进展,但骨微环境中肺腺癌细胞对血管生态位的作用及其机制仍不清楚。本研究通过探讨骨微环境驯化的肺腺癌细胞株对内皮细胞及血管生成的影响及机制,为骨微环境肿瘤细胞对血管生态位的影响提供参考。方法用骨微环境驯化的肺腺癌细胞A549-GFP-LUC-BM3(BM3)培养上清(BM3-CM)和A549-GFP-LUC(A549)培养上清(A549-CM)分别作用于人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC),用克隆形成实验、划痕实验及血管生成实验比较HUVEC增殖、迁移及血管生成能力的变化;基因富集分析(gene set enrichment analysis,GSEA)、逆转录定量聚合酶链反应(reverse transcription quantitative polymerase chain reaction,RT-qPCR)以及酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)用于检测与细胞血管生成密切相关的肝细胞生长因子(hepatocyte growth factor,HGF)的表达;进一步使用重组蛋白、中和抗体、信号通路抑制剂、免疫荧光染色(immunofluorescence staining,IF)及Westernblot等技术验证HGF的关键作用以及分子机制。结果BM3-CM比A549-CM具有更强的促HUVEC增殖、迁移及血管生成作用;生物信息学结合体外实验筛选发现分泌型蛋白HGF在BM3细胞及BM-CM中显著增高(P<0.05);BM3-CM中加入HGF中和抗体可抑制BM3-CM对HUVEC的促进作用(P<0.05),A549-CM中加入HGF重组蛋白可重现BM3-CM对HUVEC的促进作用(P<0.05);BM3细胞分泌HGF可促进HUVEC的YAP(Yes相关蛋白)活化,该促进作用可能通过激活Src,使YAP入核活化实现(P<0.05);且促YAP活化作用可被HGF中和抗体所抑制(P<0.05),A549-CM中加入HGF重组蛋白可复现BM3-CM对HUVEC中YAP的活化(P<0.05)。结论骨微环境驯化的高骨转移肺腺癌细胞BM3通过HGF/YAP轴促进HUVEC增殖、迁移及血管生成,可能对血管生态位的改变具有重要作用。Background and objective The early stages of tumor bone metastasis are closely associated with changes in the vascular niche of the bone microenvironment,and abnormal angiogenesis accelerates tumor metastasis and progression.However,the effects of lung adenocarcinoma(LUAD)cells reprogrammed by the bone microenvironment on the vascular niche within the bone microenvironment and the underlying mechanisms remain unclear.This study investigates the effects and mechanisms of LUAD cells reprogrammed by the bone microenvironment on endothelial cells and angiogenesis,providing insights into the influence of tumor cells on the vascular niche within the bone microenvironment.Methods The culture media from bone-metastatic LUAD cell A549-GFP-LUC-BM3(BM3-CM)and A549-GFP-LUC(A549-CM)were separately applied to human umbilical vein endothelial cell(HUVEC).A colony formation assay,scratch assay,and tube forma-tion assay were conducted to evaluate the proliferation,migration,and angiogenesis of HUVEC.Gene set enrichment analysis(GSEA)was conducted to identify enriched pathways,while reverse transcription quantitative polymerase chain reaction(RT-qPCR)and enzyme linked immunosorbent assay(ELISA)were performed to quantify hepatocyte growth factor(HGF),a protein that plays a crucial role in angiogenesis.Furthermore,the pivotal function of HGF and its underlying molecular mechanisms have been substantiated through the utilisation of recombinant proteins,neutralising antibodies,pathway inhibi-tors,immunofluorescence staining,and Western blot.Results BM3-CM demonstrated a more pronounced impact on the proliferation,migration,and angiogenesis of HUVEC compared to A549-CM.Bioinformatics analysis,combined with in vitro experiment,demonstrated that the secretory protein HGF was significantly elevated in BM3 cells and BM3-CM(P<0.05).The addition of HGF neutralizing antibodies to BM3-CM inhibited the promoting effect of BM3-CM on HUVEC(P<0.05),while the addition of recombinant HGF to A549-CM reproduced that promoting effect of BM3-CM on HUVEC

关 键 词：骨转移肺腺癌细胞 血管生成 HGF YAP 

分 类 号：R73[医药卫生—肿瘤]