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

Effects of 5-Aza-CdR in combination with gemcitabine on the expression of protocadherin 8 gene in pan- creatic cancer cell lines

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.