肺腺癌细胞EGFR基因过表达和突变通过介导CXCR4/CXCL12信号通路表达导致肿瘤生物学行为改变的机制研究

Mechanism of EGFR Over-expression and Mutations Leading to Biological Characteristics Changes of Human Lung Adenocarcinoma Cells through CXCR4/CXCL12 Signaling Pathway

背景与目的表皮生长因子受体(epidermal growth factor receptor,EFGR)突变与肺腺癌侵袭转移密切相关,CXCR4/CXCL12(chemokine receptor 4/chemokine ligand 12)生物学轴在肿瘤器官特异性转移中发挥重要作用,二者在肺腺癌转移过程中是否存在相互作用尚未明确。本研究旨在探索EGFR过表达和不同位点突变对肿瘤细胞增殖、迁移和侵袭等生物学行为的影响,并探讨其潜在机制。方法构建EGFR过表达、EGFR-E746-A750缺失型(DEL,19号外显子突变)、EGFR-T790M突变型(790M,20号外显子突变)、EGFR-L858R的突变型(LR,21号外显子突变)及EGFR空载质粒,应用Lipofectamine 2000转染H1299肺腺癌细胞。应用细胞克隆实验、细胞划痕实验和Transwell实验分别检测EGFR过表达和突变对H1299细胞增殖、迁移和侵袭能力的影响,并应用RT-PCR和Western blot检测CXCR4、CXCL12,以及该信号通路下游基因MMP-2和MMP-9 m RNA和蛋白表达水平。结果 EGFR过表达和EGFR-E746-A750缺失组细胞克隆形成数目分别为28±2、28.33±4.16,且细胞迁移和侵袭能力显著高于阴性对照组与空白对照组(P<0.05)。RT-PCR和Western blot实验显示EGFR过表达和EGFR-E746-A750缺失组CXCR4、CXCL12、MMP-2和MMP-9的m RNA和蛋白表达水平显著高于阴性对照组和空白对照组(P<0.05)。结论 EGFR基因过表达和19号外显子缺失突变可通过上调CXCR4/CXCL12信号通路,促进MMP-2、MMP-9表达,从而引起肺腺癌细胞的肿瘤生物学特性发生改变,并具有更强的增殖、迁移和侵袭能力。

Background and objective The epidermal growth factor receptor （EFGR） mutation was closely related to the invasion and metastasis of lung adenocarcinoma and the biological axis of CXCR4/CXCL12 （chemokine receptor 4/chemokine ligand 12） played an important role in the organ-specific metastasis of the tumor. It was a question surrounding whether there is interaction between them in the process of lung adenocarcinoma metastasis. To investigate the potential molecular mechanisms of EGFR over-expression and EFGR-mutations effects on cell proliferation, migration and invasion, we constructed EGFR over-expression and three EFGR-mutant human lung adenocarcinoma H1299 cell sublines. Methods EGFR over-expression and three EFGR-mutant （EGFR-E746-A750del, EGFR-T790M and EGFR-L858R） plasmid were designed and transfected H1299 cells with Lipofectamine 2000. H1299 cells transfected with empty vector were negative control （NC）, and H1299 cells without transfection were set as blank control （BC）. The effects of EGFR over-expression and mutations on the proliferation, migration and invasion of H1299 cells were detected by cell cloning assay, wound healing assay and Transwell assay. The mRNA and protein expression levels of MMP-2, MMP-9, CXCR4 and CXCL12 were detected by RT-PCR and Western blot. Results Compared with negative control group and blank control group, EGFR over-expression and EGFR-E746-A750 deletion have significantly higher colony formation （28±2, 28.33±4.16; respectively） （P〈0.05） and the cell migration and invasion ability were significantly increased （P〈0.05）. RT-PCR and Western blot assay showed that the mRNA and protein expression of MMP-2, MMP-9, CXCR4 and CXCL12 in EGFR over-expression and EGFR-E746-A750 deletion group were remarkably higher than that in negative control and blank control group （P〈0.05）. Conclusion EGFR over-expression and 19 exon deletion can promote the expression of MMP-2 and MMP-9 by up-regulating CXCR4/CXCL12 signaling pathway, leading to the change of tumor biological characteristics such as higher proliferation, migration and invasion ability.