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(protocadherin8,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Westernblot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(PCDH8) 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 methyla- tion 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 administra- tion and control,5-Aza-CdR together with gemcitabine can induce a stronger apoptosis signal. Differ- ent 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 effec- tively 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 gemcit- abine.
出处
《中华肝胆外科杂志》
CAS
CSCD
北大核心
2012年第7期543-547,共5页
Chinese Journal of Hepatobiliary Surgery