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背景与目的:抑癌基因家族性腺瘤样结肠息肉病易感基因(adenomatous polyposis coli,APC)启动子区的高甲基化在很多肿瘤中被发现,与这些肿瘤的发生发展相关。本实验室在肺癌患者肿瘤组织中检测到APC甲基化率达47%,为了研究其在肺癌细胞株中的甲基化情况,并进一步了解甲基化对其转录的影响,本研究检测了3株肺癌细胞的抑癌基因APC启动子甲基化状态及其对该基因转录水平的影响。方法:提取3株肺癌细胞株(肺腺癌细胞株SPC-A1、小细胞肺癌细胞株NCI-H446、大细胞肺癌细胞株NCI-H460)的DNA,以经转甲基处理和未做处理的脐带血DNA为阳性、阴性对照,亚硫酸氢盐化学修饰后,用甲基化特异性基因扩增(methylation specific-polymerase chain reaction,MSP)和甲基化基因芯片对APC基因启动子1ACpG岛甲基化进行研究,并用实时荧光定量PCR(real time-polymerase chain reaction)技术,以Sybr-GreenⅠ为荧光染料,β-actin基因为内参照,检测mRNA转录;对甲基化阳性的NCI-H460细胞,分别用1、5、10、15μmol/L的5′-杂氮-2’-脱氧胞嘧啶(5-aza-2-deoxycytidine,5-aza-dC)试剂进行脱甲基化,提取RNA,荧光定量检测其转录变化。结果:SPC-A1和NCI-H446细胞APC甲基化阴性,NCI-H460细胞APC甲基化阳性;甲基化芯片检测NCI-H460细胞在APC启动子1A5个CpG位点均存在甲基化(687、707、714、719、726),SPC-A1和NCI-H446甲基化阴性,荧光定量结果NCI-H460的APC转录较SPC-A1和NCI-H446有明显的下降,仅为二者平均的30.04%;经5-aza-dC脱甲基化作用后,NCI-H460细胞的APC表达增加了约5～10倍,其中10μmol/L浓度作用下,APC表达增加最多。结论:肺癌细胞株NCI-H460中存在APC基因高甲基化,5-aza-dC脱甲基化试剂可以激活其转录。
BACKGROUND & OBJECTIVE: Hypermethylation in the promoter region of tumor suppressor gene family adenomatous polyposis coli (APC) is found in many tumors and is associated with the development of these tumors. In our laboratory, APC methylation rate was found to be 47% in lung cancer patients. In order to study its methylation status in lung cancer cell lines and to further understand the effect of methylation on its transcription, Methylation status of tumor suppressor gene APC promoter in three lung cancer cells and its effect on the transcription level of the gene. Methods: The DNA of three lung cancer cell lines (lung adenocarcinoma cell line SPC-A1, small cell lung cancer cell line NCI-H446, large cell lung cancer cell line NCI-H460) was extracted and treated with methyltransferase and untreated Umbilical cord blood DNA was positive and negative control. After the chemical modification of bisulfite, methylation-specific polymerase chain reaction (MSP) and methylation gene chip were used to detect the APC gene promoter 1ACpG island A The methylation status of NCI-H460 cells was analyzed by real time-polymerase chain reaction (RT-PCR) with Sybr-GreenⅠ as the fluorescent dye and β-actin as the internal control. , Demethylated with 1,5,10,15 μmol / L 5-aza-2-deoxycytidine (5-aza-dC) Fluorescence quantitative detection of transcription changes. Results: APC methylation was negative in SPC-A1 and NCI-H446 cells and methylation of APC in NCI-H460 cells was detected by methylation analysis. Methylation analysis showed that methylation was found in all 5 CpG sites of APC promoter in NCI-H460 cells 687, 707, 714, 719, 726). Methylation of SPC-A1 and NCI-H446 was negative. Fluorescence quantitative results showed that the APC transcription of NCI-H460 was significantly decreased compared with that of SPC-A1 and NCI-H446 30.04%. After 5-aza-dC demethylation, the APC expression in NCI-H460 cells increased by about 5 to 10 times, and the highest APC expression was induced by 10μmol / L concentration of 5-aza-dC. CONCLUSION: APC gene hypermethylation exists in NCI-H460 lung cancer cell line, and 5-aza-dC demethylation reagent can activate its transcription.