摘要
目的探讨EGFR信号通路与人结肠癌Caco-2细胞增殖和侵袭转移的关系及其调控分子机制。方法采用MTT法和Boyden小室体外侵袭实验检测Caco-2细胞增殖和体外侵袭能力;采用RT-PCR方法检测MMP-2、MMP-9、TIMP-1、TIMP-2基因转录水平;采用W estern b lot法检测P-EGFR和P-ERK蛋白表达。结果外源性EGF(10μg/L)增加P-EGFR和P-ERK蛋白表达同时使24 h细胞生长率提高了23.35%(P<0.01),使过膜细胞数由(208±3)上升到(241±5)(P<0.01)。当用AG1478(20μmol/L)和PD98059(40μmol/L)分别阻断EGFR和ERK/MAPK后,EGF作用消失,Caco-2细胞生长明显抑制(P<0.01),但无时间效应关系;Caco-2细胞体外侵袭力明显减弱(P<0.01)。RT-PCR测定显示,EGF能增加Caco-2细胞MMP-2、MMP-9 mRNA的表达和减少TIMP-1和TIMP-2 mRNA的表达;而AG1478能逆转EGF的作用,使MMP-2、MMP-9 mRNA的表达下降,TIMP-1和TIMP-2 mRNA的表达上升,结果MMP-2/TIMP-2比值和MMP-9/TIMP-1比值均下降(P<0.01)。结论EGFR-ERK/MAPK信号通路通过改变基质金属蛋白酶和其抑制剂mRNA比值调控人结肠癌细胞侵袭转移能力。
Objective To study the effect and molecular mechanism of EGFR pathway mediating proliferation and invasion of Caco-2 cell line. Methods The tetrazolium-based colorimetric assay was used to evaluate the cell growth treated by EGF,AG1478 or PD98059. The in vitro invasion assay was used to examine the invasiveness of Caco-2 cells treated by EGF, AG1478 or PD98059. MMP-2, MMP-9, TIMP-1 and TIMP-2 mRNA of Caco-2 cells were measured by RT-PCR. Expressions of P-EGFR and P-ERK protein of Caco-2 cells were de- termined by Western blotting. Results Exogenous EGF enhanced significantly the growth and proliferation of Caco-2 cells. The growth ratio increased 23.35% （P 〈0.01 ） at 24 h. AG1478 （20 μmol/L） as EGFR inhibitor and PD98059 （40 μmol/L） as ERK inhibitor inhibited the growth and proliferation of Caco-2 cells in a time-independent manner （P 〈0.01 ）. In vitro invasion assay showed that 10 μg/L EGF increased the invasion （P 〈0.01 ） of Caco-2 cells, while both AG1478 and PD9805 inhibited EGF-induced invasion of Caco-2 cells （P 〈0.01 ）. RT-PCR assays revealed that exogenous EGF up-regulated mRNA levels of MMP-2 and MMP-9 and down-regulated mRNA levels of TIMP-1 and TIMP-2, and AG1478 and PD98059 decreased the levels of MMP-2, MMP-9 mRNA and increased the levels of TIMP-1 and TIMP-2 mRNA. The MMP-2 to TIMP-2 ratio and the MMP-9 to TIMP-1 ratio were decreased significantly by AG1478 or PD98059 （P 〈0.01 ）. Conclusion EGFR signal pathway adjusts the growth, invasion and metastasis of human colon carcinoma cells. The mechanisms may be that EGFR changes matrix metalloproteinases and its inhibitors by ERK/MAPK signal pathway.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2006年第14期1479-1482,共4页
Journal of Third Military Medical University
基金
辽宁省科技攻关课题(2003225007-3)~~
