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目的:研究hedgehog信号通路的负调控基因生长停顿特异蛋白1(growth arrest specific1,GAS1)、人类hedgehog相互作用蛋白(human hedgehog interacting protein,HHIP)和patched1(PTCH1)在早期非小细胞肺癌(NSCLC)中的甲基化状态并初步探讨此3个基因对早期NSCLC的诊断价值。方法:收集Ⅰ期NSCLC患者85例(NSCLC组)并以同期住院的23例非肿瘤性肺部疾病患者作为对照组。采用甲基化特异性聚合酶链反应(methylation-specific polymerase chain reaction,MSP)检测GAS1、HHIP和PTCH1在5个NSCLC细胞株A549、NCI-H1299、NCI-H460、LTEP-a-2、SPC-A-1中的甲基化状态及其在NSCLC组患者的肺癌组织中、对照组的肺部病变组织中的甲基化状态;分析3个基因诊断Ⅰ期NSCLC的敏感性和特异性。结果:hedgehog信号通路的3个负调控基因除在NCI-H1299中均呈去甲基化状态外,在其他4个肺癌细胞株中均有1～2个基因呈甲基化状态。GAS1、HHIP和PTCH1在NSCLC组的甲基化率高于对照组,差异有统计学意义(P<0.05)。GAS1用于临床Ⅰ期NSCLC诊断时敏感性和特异性分别为69.41%、69.57%;HHIP分别为49.41%、82.61%;PTCH1则分别为34.12%、100.0%。结论:PTCH1、HHIP和GAS1在Ⅰ期NSCLC中存在显著的异常甲基化,提示hedgehog的异常激活可能参与了NSCLC的发生和发展。对3个基因在早期NSCLC患者血浆游离DNA中甲基化状态的进一步研究可望为NSCLC的早期诊断提供新的肿瘤分子学标志物。
Objective: To investigate the effects of growth arrest specific1 (GAS1), human hedgehog interacting protein (HHIP) and patched1 (PTCH1) on hedgehog signaling pathway in early non-small cell lung cancer (NSCLC) In the methylation status and preliminary study of the three genes in the early diagnosis of NSCLC. Methods: Totally 85 patients with stage Ⅰ NSCLC (NSCLC group) were enrolled. Twenty-three patients with non-neoplastic lung disease were enrolled as control group. The methylation-specific polymerase chain reaction (MSP) was used to detect the expression of GAS1, HHIP and PTCH1 in 5 NSCLC cell lines A549, The methylation status in A-1 and its methylation status in the lung tissue of the control group in the lung cancer tissues of the NSCLC patients; The sensitivity and specificity of the 3 genes in diagnosing stage I NSCLC were analyzed. Results: The three negative regulatory genes of hedgehog signaling pathway were all methylated except NCI-H1299. One or two of the four other lung cancer cell lines were methylated. The methylation rates of GAS1, HHIP and PTCH1 in NSCLC group were higher than those in control group, with statistical significance (P <0.05). The sensitivity and specificity of GAS1 in the clinical stage Ⅰ NSCLC diagnosis were 69.41% and 69.57%, respectively; HHIP was 49.41% and 82.61% respectively; while PTCH1 was 34.12% and 100.0% respectively. Conclusion: The abnormal methylation of PTCH1, HHIP and GAS1 in stage Ⅰ NSCLC suggests that the abnormal activation of hedgehog may be involved in the occurrence and development of NSCLC. Further studies on the methylation status of the three genes in plasma free DNA in patients with early stage NSCLC are expected to provide new molecular markers of cancer for the early diagnosis of NSCLC.