【摘要】 目的 探讨Ephrin-A5(EFNA5)沉默对胰腺癌侵袭与转移能力的影响，并阐明其可能通过Pygopus 2(PYGO2)/Wnt信号通路介导的作用机制。方法 收集本院胰腺癌患者的癌组织及配对癌旁组织，采用实时荧光定量聚合酶链反应（qRT-PCR）和免疫组织化学法检测EFNA5的表达，同时分析EFNA5的表达与预后的相关性。体外实验选用高侵袭性人胰腺癌细胞系PANC-1，通过构建EFNA5沉默和PYGO2过表达细胞模型，利用划痕实验和Transwell小室实验分析其迁移与侵袭能力的变化；体内实验则通过尾静脉注射PANC-1细胞建立胰腺癌肺转移小鼠模型，观察EFNA5沉默对胰腺癌肺转移的影响，采用苏木精-伊红（H&E）染色观察肺组织微转移灶形态，蛋白免疫印迹法（WB）检测通路相关蛋白PYGO2、Wnt、β-连环蛋白（β-catenin）、增殖标志物Ki-67及侵袭相关蛋白基质金属蛋白酶-9(MMP-9)的表达水平，并通过免疫荧光实验观察β-catenin在胞浆与胞核中的定位变化。结果 EFNA5在胰腺癌组织中显著高表达，且EFNA5的表达与预后不良呈正相关。体外实验表明，沉默EFNA5可显著抑制PANC-1细胞的迁移和侵袭能力，过表达PYGO2后PANC-1细胞的迁移和侵袭能力显著恢复；体内实验显示，EFNA5沉默组小鼠肺转移结节数量明显减少，H&E染色证实微转移灶减少。Western blot结果显示，EFNA5沉默后PYGO2、Wnt、β-catenin、Ki67和MMP-9蛋白表达均下调，免疫荧光进一步表明β-catenin核转位受阻。结论 EFNA5沉默可通过抑制PYGO2/Wnt/β-catenin信号通路，降低β-catenin核定位及下游靶基因表达，进而抑制胰腺癌细胞的增殖、侵袭与转移能力。更多还原

【Abstract】 Objective To explore the effect of Ephrin-A5(ephrin-A5, EFNA5) silencing on the invasion and metastasis ability of pancreatic cancer, and to clarify its possible mechanism mediated by the Pygopus 2(PYGO2)/Wnt signaling pathway.Methods Cancer tissues and paired adjacent normal tissues from pancreatic cancer patients in our hospital were collected. The expression of EFNA5 was detected using quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry,and the correlation between EFNA5 expression and patient prognosis was analyzed. In vitro experiments utilized the highly invasive human pancreatic cancer cell line PANC-1. EFNA5-silenced and PYGO2-overexpressing PANC-1 cell models were constructed. Changes in migration and invasion abilities were analyzed using the scratch wound healing assay and Transwell chamber assay. For in vivo experiments, a mouse model of pancreatic cancer lung metastasis was established via tail vein injection of PANC-1 cells to observe the effect of EFNA5 silencing on lung metastasis. Hematoxylin-eosin(H&E) staining was used to observe the morphology of micro-metastatic foci in lung tissues. Western blot was performed to detect the expression levels of pathway-related proteins PYGO2, Wnt, β-catenin, the proliferation marker Ki67, and the invasion-related protein matrix metalloproteinase-9(MMP-9). Immunofluorescence assay was used to observe the changes in the localization of β-catenin in the cytoplasm and nucleus. Results EFNA5 was significantly highly expressed in pancreatic cancer tissues, and its expression was positively correlated with poor prognosis. In vitro experiments showed that silencing EFNA5 significantly inhibited the migration and invasion abilities of PANC-1 cells, while overexpression of PYGO2 significantly restored these abilities. In vivo experiments demonstrated that the number of lung metastatic nodules was significantly reduced in the EFNA5-silenced group, which was confirmed by H&E staining showing reduced micro-metastatic foci. Western blot results indicated that the protein expression levels of PYGO2, Wnt, β-catenin, Ki67, and MMP-9 were downregulated after EFNA5 silencing. Immunofluorescence further revealed that β-catenin nuclear translocation was blocked. Conclusion Silencing EFNA5 can inhibit the PYGO2/Wnt/β-catenin signaling pathway, reduce the nuclear localization of β-catenin and the expression of downstream target genes, thereby suppressing the proliferation, invasion,and metastasis capabilities of pancreatic cancer cells.更多还原