| Objective:The purpose of this study was to verify whether emodin can exert a certain degree of demethylation on the pancreatic cancer tumor suppressor genes P16,RASSF1 A,and ppENK,and whether the combination with 5AzA-cdR can enhance the demethylation of 5 AzA-cdR on pancreatic cancer cell tumor suppressor gen.Methods:Effects of emodin on pancreatic cancer cell proliferation were determined by using a CCK-8 kit,MSP and BSP was used to detect the methylation status of emodin,5AzA-cdR and emodin in combination with 5AzA-cdR on pancreatic cancer cell tumor suppressor gene P16,RASSF1 A,and ppENK.Dot-blot experiments verify the effects of emodin,5AzA-cdR,and emodin combined with 5AzA-cdR on the expression of pancreatic cancer Pancl cell genome 5mc.FQ-PCR and Western Blot was used to examine the mRNA and protein expression of the three tumor-suppressor genes P16,RASSF1A,ppENK and methyltransferase DNMT1,DNMT3a and DNMT3b.Results:CCK-8 confirms that emodin inhibits the growth of pancreatic cancer cell Panel in a dose-and time-dependent manner,Dot-blot results confirm that emodin combined with 5AzA-cdR can significantly inhibit the expression of 5mc in Panel cell genome.The results of MSP and BSP confirmed that the demethylation of emodin was weak,5AzA-cdR had a certain degree of demethylation,while the demethylation of emodin combined with 5AzA-cdR was more significant on pancreatic cancer cell Panel tumor suppressor gene P16,RASSF1A,and ppENK.At the same time,the results of FQ-PCR and WB confirmed that the expression of P16,RASSF1A and ppENK was significantly increased when the emodin and 5AzA-cdR were combined.The expression of DNMT1 and DNMT3a was significantly decreased compared with the control group.Conclusion:The emodin combined with 5AzA-cdR can enhance the demethylation of 5AzA-cdR on pancreatic cancer cell tumor suppressor gene P16,RASSF1A and ppENK though down-regulating the expression of methyltransferase DNMT1 and DNMT3a. |
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