熔解曲线法用于肺癌APC基因甲基化模式的研究

Investigation of Methylation Patterns of APC Gene in Lung Cancer with a Novel Fluorescence Melting Curve Analysis Assay

目的以熔解曲线法测定4株肺癌细胞株和肺癌病人APC基因启动子区的甲基化模式。方法以脐血淋巴细胞DNA及其转甲基后的DNA经化学修饰、克隆测序的质粒，作为完全非甲基化和完全甲基化标准品。设计通用引物，采用加入荧光染料SYBRGreen1的荧光定量PCR法扩增包含21个CpG位点的APC基因启动子区的目的序列，以熔解曲线法通过与完全非甲基化和完全甲基化标准品的Tm值比较，确定四株肺癌细胞株（NCI-H446，NCI-H460，SPCA1，NCI—H520）在该区段的甲基化模式，并通过克隆测序验证。同时测定两例肺癌患者癌组织中APC基因启动子区甲基化模式。结果四株肺癌细胞株中，NCI-H446，SPCA1和NCI—H520的Tm值与完全非甲基化标准品的Tm值相同，而NCI—H460的Tm值有两个，分别与完全非甲基化和完全甲基化标准品的Tm值吻合，并经克隆测序验证。两例肺癌患者癌组织的Tm值位于完全非甲基化和完全甲基化标准品的Tm值之间。结论小细胞肺癌细胞株NCI—H446，肺腺癌细胞株SPCA1和肺鳞癌细胞株NCI—H520的APC基因启动子区为完全未甲基化型，而大细胞肺癌细胞株NCI—H460APC基因启动子区甲基化模式为等位基因杂合型。两例肺癌患者癌组织中的APC基因启动子均为部分甲基化型。熔解曲线法是简单、经济和实用的甲基化模式检测方法。

Objective To identify methylation patterns in the promoter region of APC gene in lung cancer cell lines and cancer patients by fluorescence melting curve analysis assay. Methods After bisulfate treatment, DNA samples of lymphocytes from cord blood without and with trans-methyl treatment were amplified. The amplicons were then cloned into plasmid vector and employed as unmethylation and methylation controls. Universal primes were designed to amplify the target sequence in the APC gene promoter region comprising 21 CpG sites. DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye （SYBR Green I） during the dissociation stage. The methylation patterns of 4 lung cancer cell lines and lung cancer patients＇ tumor tissue cells were determined by comparison of melting temperatures （Tin） with unmethylation and methylation controls and sequencing. Results Melting curve analysis showed that three of 4 lung cancer cell lines （NCI-H446,SPCA1,NCI-H520） displayed a melting temperature 83℃ as low as the unmethylated control, while the other one （NCI-H460） displayed 2 melting peaks of 83℃ and 88℃ which were corresponding to the Tm of unmethylated and metbylated control,respectively. Sequencing reports were all in accordance with the melting curve analysis. The Tm values of two lung cancer,patients were both between the values of unmethylation and methylation controls. Conclusion The APC promoter region methylation patterns of NCI-H446 and SPCA1 and NCI-H520 are described as unmethylated alleles,while NCI-H460 cells exhibit monoallelic methylation. Two lung cancer patients＇ tumor tissue cells display partial methylation in APC promoter region. Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.