大黄素对胰腺癌细胞抑癌基因P16、RASSF1A去甲基化作用研究

Demethylation effect of emodin on tumor suppressor gene P16 and RASSF1A in Panc1 pancreatic cancer cell

目的研究大黄素对胰腺癌细胞Panc1抑癌基因P16、RASSF1A启动子区CpG岛的去甲基化作用,探讨大黄素是否可以逆转抑癌基因的甲基化状态,从而使抑癌基因重新表达生物活性,发挥抗癌活性。方法采用CCK8法检测不同浓度大黄素对胰腺癌细胞的生长情况,BSP检测不同浓度大黄素处理前后P16、RASSF1A基因甲基化状态的改变,并用RT-PCR和Western blotting分别检测两个抑癌基因以及甲基转移酶DNMT1、DNMT3a、DNMT3b在mRNA和蛋白的表达情况。结果大黄素以时间梯度和浓度梯度依赖性抑制Panc1细胞生长,BSP结果证实大黄素处理后可使P16、RASSF1A甲基化状态减弱,非甲基化状态增强,同时RT-PCR和Western blotting结果证实在Panc1中低表达或无表达的mRNA和蛋白表达增强或者重新表达。结论大黄素可以对胰腺癌细胞Panc1抑癌基因P16和RASSF1A发挥不同程度的去甲基化作用,使因启动子区甲基化而失表达的抑癌基因重新表达,大黄素抑制胰腺癌细胞生长可能和其具有对抑癌基因去甲基化作用有关,并且可推测大黄素发挥去甲基化作用和其可不同程度抑制甲基转移酶表达有一定关系。

Objective To study the demethylation effect of emodin on tumor suppressor gene P16 and RASSF1A promoter CpG Island in Pancl pancreatic cancer cell, and to find out whether emodin can reverse the methylation of cancer suppressor gene, making the tumor suppressor gene reexpress anticancer activity. Methods CCK8 was used to detect the growth of pancreatic cancer cell. Bisulfite genomic sequencing PCR （BSP） was used to detect methylation status changes of tumor suppressor gene PI6 and RASSF1A gene. While mRNA and protein production of the two tumor suppressor genes and methyltransferases DNMT1, DNMT3a and DNMT3b were evaluated by RT-PCR and Westem blotting. Results Emodin inhibited the growth of pancreatic cancer cells （Panc- 1） in a dose and time-dependent manner. BSP confirmed that the methylated status of the promoter regions of P16 and RASSF1A decreased, at the same time, the unmethylated status enhanced. The RT-PCR and Western blotting results confirmed the enhanced expression or re-expression of mRNA and protein in Pancl cells. Conclusion Emodin can exert different degrees of demethylation effect on tumor suppressor gene P16 and RASSF1A gene in pancreatic cancer cells, making the none expressed tumor suppressor gene to re-express. Emodin inhibites pancre- atic cancer cells growth, which may be related to its demethylation of tumor suppressor genes, and we can speculate that there is a relationship between the effect of emodin on demethylation and a varying degree inhibition of methyl transferase expression.