Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.
Suppression of the Epidermal Growth Factor-like Domain 7 and Inhibition of Migration and Epithelial-Mesenchymal Transition in Human Pancreatic Cancer PANC-1 Cells.
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DOI：
10.7314/apjcp.2015.16.9.4065
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发表时间：
2015
期刊：
影响因子：
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通讯作者：
Yunliang Wang;F. Dong;Jian Yang;Zhi Li;Qiaoming Zhi;Xin Zhao;Yong Yang;Dechun Li;Xiaochun She
中科院分区：
文献类型：
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作者：
Yunliang Wang;F. Dong;Jian Yang;Zhi Li;Qiaoming Zhi;Xin Zhao;Yong Yang;Dechun Li;Xiaochun She
背景表皮生长因子样结构域7（Epidermal growth factor-like domain multiple 7，EGFL 7）是一种在胚胎发育过程中由内皮细胞特异性表达的分泌蛋白，近年来被认为是肿瘤转移的关键基因。上皮间质转化（EMT）与肿瘤的进展密切相关。因此，研究EGFL 7基因表达下调后人胰腺癌细胞的迁移和EMT变化是非常重要的。材料与方法采用实时荧光定量PCR（Real-time PCR）和western blot方法检测4种胰腺癌细胞株中EGFL 7的表达，其中PANC-1细胞株EGFL 7的表达最高。然后，将使用质粒载体转染EGFL 7的小干扰RNA（siRNA）的PANC-1细胞命名为si-PANC-1，而将转染阴性对照质粒载体的PANC-1细胞命名为NC-PANC-1。Transwell法检测PANC-1细胞的迁移能力。采用Real-time PCR和western blotting分别检测PANC-1、NC-PANC-1和si-PANC-1细胞中EGFL 7基因、EMT标志物E-Cadherin、N-Cadherin、Vimentin、Fibronectin和转录因子snail、slug的表达变化。结果质粒转染成功后，si-PANC-1组EGFL 7基因被RNA干扰显著下调。与PANC-1组和NC-PANC-1组相比，细胞迁移能力明显降低。同时，si-PANC-1组上皮细胞表型标志物E-Cadherin表达增加，间质细胞表型标志物N-Cadherin、Vimentin、Fibronectin表达明显减少，提示EMT逆转。此外，转录因子snail和slug在RNA干扰后显著降低。结论目前的研究表明，高表达的EGFL 7促进PANC-1细胞的迁移，并通过转录因子snail和slug诱导EMT，这一问题需要进一步研究证实。
BACKGROUND Epidermal growth factor-like domain multiple 7 (EGFL7), a secreted protein specifically expressed by endothelial cells during embryogenesis, recently was identified as a critical gene in tumor metastasis. Epithelial-mesenchymal transition (EMT) was found to be closely related with tumor progression. Accordingly, it is important to investigate the migration and EMT change after knock-down of EGFL7 gene expression in human pancreatic cancer cells. MATERIALS AND METHODS EGFL7 expression was firstly testified in 4 pancreatic cancer cell lines by real-time polymerase chain reaction (Real-time PCR) and western blot, and the highest expression of EGFL7 was found in PANC-1 cell line. Then, PANC-1 cells transfected with small interference RNA (siRNA) of EGFL7 using plasmid vector were named si-PANC-1, while transfected with negative control plasmid vector were called NC-PANC-1. Transwell assay was used to analyze the migration of PANC-1 cells. Real-time PCR and western blotting were used to detect the expression change of EGFL7 gene, EMT markers like E-Cadherin, N-Cadherin, Vimentin, Fibronectin and transcription factors like snail, slug in PANC-1, NC- PANC-1, and si-PANC-1 cells, respectively. RESULTS After successful plasmid transfection, EGFL7 gene were dramatically knock-down by RNA interference in si-PANC-1 group. Meanwhile, migration ability decreased significantly, compared with PANC-1 and NC-PANC-1 group. Meanwhile, the expression of epithelial phenotype marker E-Cadherin increased and that of mesenchymal phenotype markers N-Cadherin, Vimentin, Fibronectin dramatically decreased in si-PANC-1 group, indicating a reversion of EMT. Also, transcription factors snail and slug decreased significantly after RNA interference. CONCLUSIONS Current study suggested that highly-expressed EGFL7 promotes migration of PANC-1 cells and acts through transcription factors snail and slug to induce EMT, and further study is needed to confirm this issue.