循环miR-21与表皮生长因子受体的相关性研究

miRNAs are a class of short noncoding RNAs and involved in the development, progression and metastasis of cancer. miRNAs can stably exist in the blood. Circulating miRNAs were seen as an important non-invasive means for detecting tumor. miR-21 was up-regulated in lung cancer and related to chemotherapeutical sensitivity. Epidermal growth factor (EGFR), the therapeutic target of lung cancer, was over-expressed or mutated in most lung cancer (especially non-small cell lung cancer). NSCLC patients with EGFR mutations were sensitivity to EGFR tyrosine kinase inhibitors that had anti-tumor effect by blocking EGFR signal pathway and inducing tumor cell apoptosis. EGFR mutations were more frequently in female never-smoker adenocarcinoma patients in East Asia. Therefore, the study aimed to explore in the following aspects: (1) the correlation between the expression level of circulating miR-21 and EGFR mutation; (2) the correlation between the expression level of circulating miR-21 and the protein concentrations of EGFR in peripheral blood. It was hopeful to investigate the relation between circulating miR-21 and EGFR, so as to identify a new type of diagnostic marker, improve the choice of targeted therapy and better supply the basis for clinical treatment.

miRNA是一类非编码小分子RNA，其参与肿瘤的发生、发展及侵袭转移等。miRNA能够非常稳定的存在于血液循环中，检测循环miRNA的表达情况可以作为一种重要的无创性检测肿瘤的手段。miR-21在肺癌中表达上调，还与化疗敏感性相关。表皮生长因子受体（EGFR）在大多数肺癌中过表达或发生突变，其是肺癌的重要治疗靶标。EGFR突变型的NSCLC患者对EGFR酪氨酸激酶抑制剂（ TKI）敏感，而EGFR-TKI通过阻断EGFR信号通路和诱导肿瘤细胞凋亡发挥抗肿瘤作用。EGFR突变多见于东亚人种、女性、非吸烟的腺癌患者。因此，本课题拟研究（1）循环miR-21的表达水平和EGFR突变状态的相关性；（2）循环miR-21的表达水平和外周血EGFR蛋白浓度的相关性。希望通过探索循环miR-21和?和EGFR之间的关系，从而鉴定肺癌的新型诊断性标记物，并完善靶向治疗的选择，更好地为临床治疗提供依据。

本课题研究了非小细胞肺癌（NSCLC）患者外周血表皮生长因子受体（EGFR）蛋白浓度、EGFR基因突变状态及循环miR-21的表达水平。应用酶联接免疫吸附剂测定(ELISA)方法测定正常对照和NSCLC患者的EGFR蛋白表达水平。结果显示，NSCLC患者的EGFR蛋白表达水平显著高于正常对照，两者间的差异具有统计学意义(P<0.05)。使用扩增阻遏突变系统（ARMS）法检测241例NSCLC患者的EGFR基因突变状态。在241例中，132例检测到EGFR基因突变，而109例未检测到EGFR基因突变。EGFR的基因突变包括第18号外显子点突变（G719X）、第19号外显子缺失突变（DEL突变）、第20号外显子点突变（S768I）和第21号外显子点突变（L858R）。这四种类型的基因突变均与抗EGFR酪氨酸激酶抑制剂的治疗敏感相关。研究发现，外周血EGFR蛋白浓度与EGFR基因突变状态不相关。利用实时定量聚合酶链式反应（real-time PCR）法对NSCLC 患者和正常对照中的循环miR-21 的表达水平进行了检测。结果表明，与正常对照相比，NSCLC患者的miR-21表达水平增高。NSCLC患者中的miR-21表达水平与临床分期、病理类型、淋巴结转移(P值均<0.05)有关,表明miR-21与NSCLC的发生、发展及侵袭转移等。同时，我们还收集了肿瘤患者及正常对照的外周血标本以建立标本库，为后续研究做准备。