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目的调查上皮-间质转化(epithelial-mesenchymal transition,EMT)在非小细胞肺癌(non-small cell lungcancer,NSCLC)患者接受吉非替尼(Gefitinib)治疗反应性中的作用及机制。方法突变富集PCR法检测NSCLC患者EGFR突变状况;免疫组化法检测癌组织中上皮钙粘蛋白(E-cadherin)和纤维连接蛋白(Fibronectin)的表达情况,探讨EMT与表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor tyrosine kinase inhibitors,EGFR-TKIs)治疗敏感性的关系。体外选取人肺腺癌细胞系PC9细胞,经TGF-β1反复处理4周,观察细胞形态学变化;MTT检测TGF-β1处理后细胞对Gefitinib敏感性的影响;Western blot验证EMT相关标记蛋白表达变化并检测EGFR信号通路下游蛋白的变化。结果 43例NSCLC标本中,EGFR 19、21外显子突变率为58.14%(25/43)。具有EGFR基因突变的肿瘤E-cadherin的表达水平显著高于EGFR野生型(70.00%vs 30.00%,P<0.05)。接受Gefitinib总体有效率为46.51%(20/43),具有E-cadherin阳性表达的患者治疗反应性明显好于Fibronectin阳性的患者(65.00%vs 30.43%,P<0.05)。TGF-β1可诱导PC9细胞向间质型细胞形态转化,上调Fibronectin的表达;与亲本PC9细胞相比,TGF-β1处理的细胞对Gefitinib的敏感性下降(P<0.05);这种敏感性的下降伴随着AKT和STAT3的持续活化。结论 EMT在EGFR-TKI耐药中发挥着重要作用,TGF-β1诱导的EMT可影响PC9细胞对Gefitinib的敏感性,这种效应可能是通过持续活化AKT和STAT3而发挥作用。
Objective To investigate the role and mechanism of epithelial-mesenchymal transition (EMT) in the response to Gefitinib in non-small cell lung cancer (NSCLC) patients. Methods Mutations and enrichment PCR were used to detect the mutation of EGFR in NSCLC patients. The expression of E-cadherin and fibronectin in cancer tissues was detected by immunohistochemistry. The relationship between EMT and epidermal growth factor receptor Relationship between sensitivity to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) treatment. In vitro, the human lung adenocarcinoma cell line PC9 was selected and treated with TGF-β1 for 4 weeks to observe the morphological changes. The effect of TGF-β1 on Gefitinib sensitivity was examined by MTT assay. The expression of EMT-related marker protein was detected by Western blot And detect the changes of the downstream protein of EGFR signaling pathway. Results In 43 NSCLC specimens, the exon mutation rate of EGFR 19 and 21 was 58.14% (25/43). The expression of E-cadherin in tumor with EGFR gene mutation was significantly higher than that in EGFR wild type (70.00% vs 30.00%, P <0.05). The overall response rate to Gefitinib was 46.51% (20/43). The response to treatment with E-cadherin was significantly better in patients with positive Fibronectin (65.00% vs 30.43%, P <0.05). TGF-β1 induced the transformation of PC9 cells into mesenchymal cells and up-regulated the expression of Fibronectin. Compared with parental PC9 cells, the sensitivity of TGF-β1-treated cells to Gefitinib decreased (P <0.05). This sensitivity The decline is accompanied by the sustained activation of AKT and STAT3. Conclusion EMT plays an important role in EGFR-TKI resistance. EMT induced by TGF-β1 may affect the sensitivity of PC9 cells to Gefitinib. This effect may be through sustained activation of AKT and STAT3.