Src酪氨酸激酶在人胃癌细胞转移中的作用和机制

Role and mechanism of Src tyrosine kinase on human stomach cancer metastasis

背景与目的:Src酪氨酸激酶的活化在胃癌浸润和转移中发挥了重要的作用。本研究旨在探讨Src酪氨酸激酶对人胃癌细胞E-钙黏着蛋白(E-cadherin)和基质金属蛋白酶(matrix metalloproteinase,MMP)-2、9表达、血管内皮生长因子(vascular endothelial growth factor,VEGF)分泌、转录因子NF-κB和AP-1表达以及胃癌细胞体外侵袭能力的影响。方法:使用Src酪氨酸激酶抑制剂4-苯胺喹唑啉(3和10μmol/L)后,应用Western blot法检测人胃癌SGC7901细胞中E-cadherin、MMP-2和MMP-9蛋白表达变化;应用实时PCR(real-time PCR)检测细胞中E-cadherin、MMP-2和MMP-9的mRNA表达变化;应用ELISA法检测细胞培养上清液中VEGF表达变化;应用双荧光报告基因法检测NF-κB和AP-1转录活性;应用Transwell法检测肿瘤细胞侵袭能力变化。结果:当Src酪氨酸激酶抑制剂4-苯胺喹唑啉浓度为3和10μmol/L时,SGC7901细胞中E-cadherin蛋白表达量显著增加,E-cadherin的mRNA表达是对照组的263.8%(P<0.05)和403.3%(P<0.05);MMP-2和MMP-9的蛋白表达显著降低,MMP-2的mRNA表达是对照组的28.3%(P<0.05)和16.7%(P<0.05),MMP-9的mRNA表达是对照组的23.6%(P<0.05)和13.3%(P<0.05);VEGF含量是对照组的62.6%(P<0.05)和38.7%(P<0.05);AP-1转录活性是对照组的54.4%(P<0.05)和18%(P<0.05),NF-κB转录活性是对照组的38.3%(P<0.05)和11.5%(P<0.05);胃癌细胞侵袭能力是对照组的43.3%(P<0.05)和28.2%(P<0.05),呈现明显的剂量依赖性抑制作用。结论:Src酪氨酸激酶活化与人胃癌SGC7901细胞体外转移能力密切相关,其机制可能与肿瘤转移相关基因表达有关。

Background and purpose： The Src tyrosine kinase activation plays an important role in stomach cancer invasion and metastasis. The aim of this research was to evaluate the effect of Src tyrosine kinase inhibition on the expression of E-cadherin and matrix metalloproteinase-2, -9 （MMP-2, MMP-9）, expression of vascular endothelial growth factor （VEGF）, transcription activity of NF-r,B, AP-1 and tumor invasion potential in vitro in human stomach cancer SGC7901 cell line. Methods： 4-anilinoquirazoline was used to inhibit Src tyrosine kinase in human stomach cancer SGC7901 cell line, and then Western blot assay was used to examine the expression of E-cadherin and MMP-2 and MMP-9 at protein level; real-time PCR was used to examine the expression of E-cadherin and MMP-2 and MMP- 9 at mRNA level; ELISA assay was used to examine the expression of VEGF in culture media supernatant; reporter genes assay was used to examine the expression of NF-κ3 and AP-1 in transcriptional level; Transwell assay was used to examine the invasion potential of tumor cells in vitro. Results： Src tyrosine kinase inhibitor 4-anilinoquirazoline at 3 and 10 μmol/L obviously increased the expression of E-cadherin at protein and mRNA levels （P〈0.05）, and decreased the MMP-2 and MMP-9 at protein and mRNA levels （P〈0.05）, also decreased the expression of VEGF, NF-nB and AP-1 （P〈0.05） and the invasion potential of SGC7901 cells in vitro （P〈0.05）. The suppression by Src tyrosine kinaseinhibitor was presented in a dose-dependent manner. Conclusion： Src tyrosine kinase activity is related with the metastasis potential of SGC7901 cell line, and the possible mechanism is the change of tumor metastasis-related genes expression.