整合素α5β1和细胞外信号调节激酶信号传导通路在非小细胞肺癌中的作用及其相关性研究

Effect of α5β1 Integrin and ERK Signaling Pathway on Non-small Cell Lung Cancer

目的:研究整合素α5β1及磷酸化ERK1/2(p-ERK1/2)在非小细胞肺癌(NSCLC)组织中的表达及整合素α5β1和ERK1/2信号通路在人肺癌A549细胞生长和迁移中的作用。方法:采用免疫组织化学法和免疫印迹(Western blot)法检测41例NSCLC组织和15例正常肺组织中整合素α5β1及p-ERK1/2的表达并分析两者的相关性,体外培养肺癌A549细胞,以Anti-α5β1或ERK激酶抑制剂PD98059作为工具药物,采用四甲基偶氮唑盐比色(MTT)法和Annexin-V FITC PI双染色法检测A549增殖和凋亡,采用Western blot检测A549中MMP-9蛋白水平。采用逆转录-聚合酶链反应(RT-PCR)和Western blot检测Anti-α5β1处理后A549中ERK1/2蛋白和mRNA表达水平的变化。结果:人NSCLC组织中整合素α5β1及p-ERK1/2阳性表达率(分别为58.53%,65.52%)明显高于正常肺组织(分别为26.67%,20.00%)(P<0.01)。整合素α5β1与p-ERK1/2表达均与病理分级(分别为P<0.001,P=0.030)、淋巴结转移(分别为P=0.014,P<0.001)、临床分期(分别为P=0.027,P=0.038)相关。整合素α5β1与p-ERK1/2表达具有相关性(r=0.375,P<0.05)。经Anti-α5β1或PD98059处理后,A549细胞增殖效应和早期凋亡细胞百分率增加,MMP-9的表达均明显减弱。经Anti-α5β1处理后A549细胞中ERK的mtRNA和蛋白表达受到抑制,ERK1/2的磷酸化水平显著减少。结论:整合素α5β1及p-FRK1/2在人NSCLC组织中高表达,Anti-α5β1在下调了细胞内ERK的mRNA和蛋白磷酸化的同时,可以明显抑制A549细胞的增殖和MMP-9蛋白表达,表明整合素α5β1可能介导ERK1/2信号转导通路参与了非小细胞肺癌中的异常增殖和迁移的调控。

Objective： To study the expression of α5131 integrin and phosphorylated ERK1/2（ p-ERK1/2 ） in non-small cell lung cancer （ NSCLC ）, and the effect of α5131 integrin and ERK signal transduction pathway on proliferation and migration αA549 cells. Method： The expression of α5131 integrin and p-ERK1/2 in 41 specimens of NSCLC, and 15 controled specimens were determined by immunohistochemical assay and Western blot analysis. The correlation between the results of two methods was then analyzed. A549 cells were cultured in vitro, with anti-ct5131 and ERK inhibitor PD98059 as the tool drugs. The proliferation and apoptosis of the A549 cells were measured with the MTT assay and Annexin-V FITC PI double staining, respectively. The protein expression level of MMP-9 was measured with Western blot analysis. The protein expression levels of ERK1/2 were detected by Western blot analysis and the ex- pression levels of ERK1/2 mRNA was measured by RT-PCR. Result： The expression of α5131 integrin and p-ERK1/2 was significantly higher in NSCLC （ 58.53% and 65.52%, respectively ） compared with that in the normal lung tissue （ 26.67% and 20.00% respective- ly ）. There was also a positive correlation of ct5131 integrin and p-ERK1/2 expression with pathological characteristics （ P- 0.000, P = 0.030 ）, lymph node metastasis （ P = 0.014, P- 0.000 ）, and clinical stage （ P = 0.027, P = 0.038 ）. The expression of u5131 integrin was positively correlated with p-ERK1/2 expression （ r = 0.375, P 〈 0.05 ）. The anti-α5131 and ERK inhibitor PD98059 could inhibit the proliferation of A549 cells, resulting in an increase of the percentage in the early apoptotic cells, and down-regulated the expression of MMP-9. In addition, the anti-α5131 could inhibit the phosphorylated ratio of ERK by down-regulating the expression of ERK mRNA and the relative proteins. Conclusion： The positive rates of α5131 integrin and p-ERK1/2 are higher in NSCLC than in the normal lung tissue. Anti-α5131 can down-regulate the expression of ERK mRNA and the phosphorylated ratio, and can inhibit the proliferation of A549 cells and down-regulate the expression of MMP-9. This indicates that the α5131 integrin-mediated ERK signal transduction pathway might lead to the abnormal proliferation and migration of A549 cells.