大黄素联合5AzA-cdR对胰腺癌抑癌基因p16、RASSF1A去甲基化作用研究

Emodin enhances 5AzA-cdR-induced demethylation of P16 and RASSF1A genes in pancreatic cancer cells

目的研究大黄素是否可增强5AzA-cdR对胰腺癌Panc1细胞抑癌基因p16、RASSF1A的去甲基化作用。方法采用细胞增殖实验检测不同浓度大黄素对Panc1细胞的生长抑制情况,焦磷酸盐测序PCR(BSP)分别检测大黄素、5AzA-cdR及大黄素联合5AzA-cdR对Panc1细胞抑癌基因p16、RASSF1A甲基化状态的影响,并用荧光定量PCR(FQ-PCR)和Western blot分别检测p16、RASSF1A及甲基转移酶DNMT1、DNMT3a在mRNA和蛋白水平的表达情况。结果大黄素以时间和浓度梯度依赖性抑制Panc1细胞生长。BSP结果显示大黄素具有微弱的去甲基化作用,5AzA-cdR具有-定程度的去甲基化作用,当两者联用时,去甲基化作用更加显著;FQ-PCR和Western blot结果显示大黄素与5AzA-cdR联用时,p16、RASSF1A的表达水平均较空白对照明显增高(均P<0.05),DNMT1、DNMT3a的表达水平均较空白对照明显降低(均P<0.05)。结论大黄素与5AzA-cdR联用可通过降低DNMT1和DNMT3a的表达水平来增强5AzA-cdR对胰腺癌抑癌基因p16、RASSF1A的去甲基化作用。

Objective To investigate the effects of emodin on 5AzA-cdR-induced demethylation of tumor suppressor genes P16 and RASSF1A in pancreatic cancer cells, Methods Cultured pancreatic cancer Pancl cells were treated with emodin, 5AzA-cdR or emodin plus 5AzA-cdR, respectively. Cell proliferation was determined by CCK-8 kit; methylation of p16 and RASSF1A genes in Pancl cells was detected by BSP method. The mRNA and protein expressions of p16, RASSF1A and methyltransferase DNMT1 and DNMT3a in Pancl cells were examined by FQ - PCR and Western blot, respectively. Results Emodin inhibited the growth of pancreatic cancer Pancl cells in a dose- and time-dependent manner. BSP results showed that the demethylation effect of emodin was weak, 5AzA-cdR had a moderate effect on demethylation, while the demethylation of emodin combined with 5AzA-cdR was more significant. FQ-PCR and WB confirmed that the expression of P16 and RASSF1A increased（P〈0.05） and the expression of DNMT1 and DNMT3a decrease in combination group compared to control group（P〈 0.05）. Conclusion Emodin combined with 5AzA-cdR can enhance the 5AzA-cdR-induced demethylation of P16 and RASSF1A genes in pancreatic cancer cells though down-regulating the expression of methyltransferase DNMT1 and DNMT3a.