摘要
目的探讨EGFR基因启动子甲基化水平与人非小细胞肺癌(NSCLC)细胞株对吉非替尼敏感性之间的相关性。方法用不同浓度吉非替尼分别作用于NSCLC细胞株HCC827、H1650、H1975、H358、H1299、A549后,CCK-8法检测细胞增殖抑制率,DNA直接测序法、实时荧光定量PCR法、免疫印迹法和甲基化特异性PCR法分别检测上述NSCLC细胞株EG-FR基因突变、EGFR mRNA、EGFR蛋白表达和启动子区甲基化状态。结果 CCK-8法检测结果显示,19外显子缺失突变的HCC827细胞对吉非替尼最敏感,而同为19外显子缺失突变的H1650细胞对吉非替尼不敏感;野生型的H358细胞对吉非替尼中度敏感,其敏感性甚至超过19外显子突变的H1650细胞,而同为EGFR野生型的H1299、A549细胞对吉非替尼敏感性较差。吉非替尼处理72h后,HCC827细胞与H358细胞相比、HCC827细胞和H358细胞与其他4株细胞相比,IC50值均有显著性差异(P<0.05)。HCC827细胞EGFR启动子为未甲基化状态,其EGFR蛋白和mRNA表达最高;H358细胞为部分甲基化,其EGFR蛋白和mRNA为中等表达;其他4个细胞株均为高甲基化状态,EGFR蛋白和mRNA呈低表达;HCC827细胞的EGFR表达水平较H358细胞高,HCC827和H358细胞的EGFR表达较其他4个细胞株高,差异均有统计学意义(P<0.05)。结论EGFR基因启动子区高甲基化可能下调EGFR基因的表达水平,从而降低NSCLC对吉非替尼的敏感性;对该基因的甲基化检测可能对预测吉非替尼治疗NSCLC疗效有一定的临床指导意义。
Objective To examine the relationship between EGFR gene promoter methylation and the sensitivity of gefitinib in non-small cell lung cancer(NSCLC) cell lines. Methods HCC827, H1650, H1975, H358, H1299 and A549 cells were treated with different dose of gefitinib separately. Cell counting kit-8 (CCK-8) assay was used to determine the cell proliferation inhibition rate. The protein and mRNA expression levels of EGFR were detected by Western blotting and RT-PCR methods. The methylation of EGFR gene promoter region was examined by methylation-specific PCR. Results EGFR mutation status : HCC827 and H1650 ceils were exon 19 deletions; H1975 cells were exon 21 L858R point and exon 20 T790M insertion mutations,and H358,H1299,A549 were all wide type ceils. According to the 50% inhibition concentration( ICs0 ), the HCC827 was most sensitive to genfitinib and H538 was moderately sensitive, while H1650 was non-sensitive. H1299 and A549 were less sensitive to genfitinib. The expression of EGFR pro- tein and mRNA were relatively higher in HCC827 and H358 cells comparing with other four cell lines ( P 〈 0. 05 ). EGFR gene promot- er region was unmethylated in HCC827 cells and partly methylated in H358 cells ,while in other four cells were all methylated. Conclu- sion EGFR gene promoter methylation may contribute to the downregulation of EGFR gene and consequently affect the sensitivity of gefitinib in NSCLC cell lines. Analysis of methylation status of EGFR gene promoter may have definite value in predicting the therapeu- tic response of gefitinib.
出处
《临床肿瘤学杂志》
CAS
2012年第9期769-774,共6页
Chinese Clinical Oncology
基金
江苏省科技厅科研基金资助项目(BK2009446)
吴阶平医学基金资助项目(320.6700.09050)