摘要
目的:探讨EGFR信号通路对人黑色素瘤A375细胞增殖、粘附和侵袭的影响及其分子机制。方法:用稀释克隆技术筛选EGFR高表达细胞株;采用MTT法检测A375细胞增殖和粘附情况;利用Boyden小室模型检测A375细胞体外侵袭力;WesternBlot法检测P-EGFR和P-AKT蛋白表达;半定量RT-PCR检测MMP-2,MMP-9,TIMP-1和TIMP-2mRNA表达。结果:EGF(10ng/ml)作用A375细胞24h时使P-EGFR和P-AKT蛋白表达明显升高,同时A375细胞对Matrigel基质胶的粘附力和体外侵袭力均明显提高(P<0.05),但作用时间达72h时才表现出促细胞增殖作用;AG1478(20!mol/L)和LY290042(10!mol/L)分别封闭EGFR和PI3K活性后均能阻止EGF的作用,并以时间依赖方式抑制A375细胞生长(P<0.01),降低细胞粘附力和体外侵袭力(P<0.01)。EGF具有上调MMP-2,MMP-9和下调TIMP-1,TIMP-2mRNA表达水平作用,而AG1478能下调MMP-2,MMP-9和上调TIMP-1mRNA表达水平,但对TIMP-2mRNA表达改变不明显,结果使MMP-2/TIMP-2和MMP-9/TIMP-1mRNA比值均明显下降。结论:EGFR—PI3K/AKT信号通路参与调节黑色素瘤侵袭转移过程,MMP-2/TIMP-2和MMP-9/TIMP-1mRNA比值变化可能发挥重要作用。
Objective: To study the effect and mechanism of EGFR signaling pathway on proliferation, adhesion and invasion of A375 cells. Methods: High EGFR expression of A375 cells of human melanoma was found and cultured by diluted clone assay and cell culture technique. The cell growth was analyzed using MTT assay. The cell adhesion and invasion to ECM were measured by MTT and Boyden chamber method. Expression levels of P-EGFR and P-AKT proteins were determined using Western blot. Expression levels of MMP-2, MMP-9, TIMP-1 and of TIMP-2 mRNA were determined by RT-PCR. Results: Exogenous EGF could up-regulate P-EGFR and P-AKT at 24h, but had no further effect on the growth and proliferation of A375 cells until 72h. Meanwhile significant enhancement of adhesion and invasion to ECM was observed at 24h of EGF addition in contrast to the control (P〈 0.05). AG1478 (20μmol/L) or LY290042 (10μmol/L) could abrogate EGF effects. They could inhibit the growth and proliferation of A375 cells in a time-dependent manner (P〈0.01) and reduce the cell adhesion and cells through Matrigel (P〈0.01). EGF increased the levels of mRNA of MMP-2 and MMP-9 and reduced the levels of mRNA of TIMP-1 and TIMP-2. AG1478 could reduce the levels of mRNA of MMP-2 and MMP-9 and increased the levels of mRNA of TIMP-1, but TIMP-2 was not changed, so that ratios of MMP-2/TIMP-2 mRNA and MMP-9/TIMP-1 mRNA were significantly reduced. Conclusions: EGFR-PI3K signaling pathway mediates invasion and metastasis of human melanoma. The ratios of MMP-2/TIMP-2 mRNA and MMP-9/TIMP-1 mRNA might be playing important roles in promoting or inhibiting metastasis of human melanoma.
出处
《中国肿瘤临床》
CAS
CSCD
北大核心
2006年第7期361-366,共6页
Chinese Journal of Clinical Oncology
基金
辽宁省科技攻关基金资助(编号:2003225007-3)