5-杂氮脱氧胞嘧啶核苷联合吉西他滨对胰腺癌细胞株原钙黏蛋白8基因表达的影响

摘要

目的 研究5-杂氮脱氧胞嘧啶核苷(5-Aza-CdR)对胰腺癌细胞株Capan-2原钙黏蛋白8(protocadherin 8,PCDH8)基因DNA启动子甲基化的转录调控,以及联合吉西他滨对癌细胞生长抑制与凋亡的影响.方法 采用MTT法、流式细胞术检测5-Aza-CdR及联合吉西他滨对Capan-2细胞的抑制率与凋亡率.应用甲基化特异性PCR(MSP)、RT-PCR和Western blot的方法检测不同浓度5-Aza-CdR处理细胞前后PCDH8基因的甲基化状态、mRNA表达水平和蛋白表达水平.结果 5-Aza-CdR能使胰腺癌细胞Capan-2增长速度减慢,呈浓度依赖性,联合吉西他滨显著抑制细胞生长；5-Aza-CdR联合吉西他滨与单药组、对照组相比,可显著诱导细胞凋亡.不同浓度的5-Aza-CdR可逆转PCDH8基因在胰腺癌细胞Capan-2的甲基化,恢复mRNA表达水平和蛋白表达水平,并呈一定的浓度正相关.结论 5-Aza-CdR可有效逆转PCDH8基因在胰腺癌细胞株Capan-2的高甲基化状态,解除DNA高甲基化导致的基因沉默,重新诱导基因mRNA转录和蛋白表达,同时可抑制胰腺癌细胞生长,与吉西他滨有协同抗肿瘤作用.%Objective To investigate the effects of 5-Aza-CdR on the transcriptional regulation through methylation of the DNA promoter protocadherin 8(PCDHg) gene in pancreatic cancer cell line Capan-2.The Capan-2 retardation in growth rate and apoptosis were assessed in when administered 5-Aza-CdR and the chemotherapy agent,gemcitabine.Methods MTT and flow cytometry were used to analyze the cell growth inhibition and apoptosis when treated with 5-Aza-CdR or in combination with gemcitabine.Methylation-specific PCR,RT-PCR and western blot were performed to detect methylation state,mRNA and protein respectively of PCDH8 gene in 5-Aza-CdR-treated Capan-2cells.Results Capan-2 cells treated with 5-Aza-CdR showed a slower growth rate,and a significant growth inhibition when given both 5-Aza-CdR in combination with gemcitabine.Compared with single drug administration and control,5-Aza-CdR together with gemcitabine can induce a stronger apoptosis signal.Different concentrations 5-Aza-CdR of were able to reverse methylation,restore mRNA and protein levels of PCDH8 in Capan-2.Conclusion 5 Aza-CdR may demethylate the PCDH8 gene,which would effectively remove the gene silencing caused by high methylation,and thus induce gene mRNA transcription and protein expression to inhibit cell growth and have collaborative antitumor functions with gemcitabine.