Yue B, Ren Q-X, Su T, Wang L-N, Zhang L
Department of Spine, the Affiliated Hospital of Qingdao University, Qingdao, China.
Eur Rev Med Pharmacol Sci. 2014;18(18):2640-7.
ERK5 is over expressed in a many of human cancers and this overexpression has been associated with metastasis and invasion. Furthermore, ERK5 silencing inhibits aggressive phenotypes of cancer cells. However, mechanisms by which ERK5 regulates tumour progression or metastasis have not been elucidated. In this study, using human osteosarcoma cell lines U2OS as a model, we explored the involvement of ERK5 silencing on invasiveness of U2OS cells.
ERK5 siRNA targeting ERK5 was stably transfected into the human osteosarcoma cell lines U2OS. ERK5 knocked-down U2OS cells was then transfected with Slug cDNA or MMP-9 cDNA plasmid to re-express Slug or MMP-9. Cell proliferation was detected by MTT assay. Cell invasion and metastasis was detected by Matrigel invasion and wound healing assay. An orthotopic nude mouse model of U2OS was applied for in vivo lung metastasis experiments. ERK5, Slug, MMP-9 and E-cadherin were analyzed by real-time PCR, and Western blotting.
ERK5 silencing by siRNA in U2OS cells decreased Slug and MMP-9 expression. Compared with the vector-transfected cells, ERK5 knocked-down cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of Slug or MMP-9 in ERK5 knocked-down cells restored the invasive phenotypes. We also discovered that Re-expression of Slug in ERK5 knocked-down cells restored the MMP-9 expression, and re-expression of MMP-9 in ERK5 knocked-down cells did not affect Slug and ERK5 expression.
Our data suggest that ERK5 knockdown inhibits aggressive behaviour of human U2OS cells through modulating Slug signaling and MMP-9 expression.
ERK5在多种人类癌症中过度表达，这种过度表达与转移和侵袭有关。此外，ERK5沉默可抑制癌细胞的侵袭性表型。然而，ERK5调节肿瘤进展或转移的机制尚未阐明。在本研究中，我们以人骨肉瘤细胞系U2OS为模型，探讨ERK5沉默对U2OS细胞侵袭性的影响。
将靶向ERK5的ERK5 siRNA稳定转染到人骨肉瘤细胞系U2OS中。然后用Slug cDNA或MMP-9 cDNA质粒转染ERK5敲低的U2OS细胞，以重新表达Slug或MMP-9。通过MTT法检测细胞增殖。通过基质胶侵袭和伤口愈合试验检测细胞侵袭和转移。应用U2OS原位裸鼠模型进行体内肺转移实验。通过实时PCR和蛋白质印迹法分析ERK5、Slug、MMP-9和E-钙黏蛋白。
U2OS细胞中通过siRNA沉默ERK5可降低Slug和MMP-9的表达。与载体转染细胞相比，ERK5敲低的细胞在体外迁移和侵袭减少，在实验性转移中的转移潜力也降低。在ERK5敲低的细胞中重新表达Slug或MMP-9可恢复侵袭性表型。我们还发现，在ERK5敲低的细胞中重新表达Slug可恢复MMP-9的表达，而在ERK5敲低的细胞中重新表达MMP-9不影响Slug和ERK5的表达。
我们的数据表明，ERK5敲低通过调节Slug信号和MMP-9表达来抑制人U2OS细胞的侵袭性行为。
