Study On Demethylation Of P16^Ink4aGene In ALT Tumor Cells By Puerh And An Alkaloids Extracted From Buxaceae

Objects We did some experiments to investigate the effect of an alkaloids extracted from Buxaceae and puerh on demethylation and activation transcription of p16Ink4a gene in ALT tumor cells, whose promoter region of p16Ink4a gene was hypermethylation, and to further study their potential mechanism of demethylation, which can provide a theoretical basis for puerh and Buxaceae as two kinds of adjunctive treatments of some cancers.Methods Cell morphology detection was used to observe the morphological changes of the cells which this kind of alkaloids called KBA02and puerh treated before and after. Crystal violet staining was used to find the differences in the proliferation of the cells which was treated with different concentration of KBA02and puerh. BSP(bisuLfate sequencing PCR) was used to detect the methylation status of promoter region of p16Ink4a gene after ALT tumor cells had been treated with KBA02and puerh. The mRNA of p16M4a gene were measured by RT-PCR. The protein of p16Ink4a was detected by Western Blotting. The above methods were used5-Aza-CdR as a positive control.Results The promoter region of p16Ink4a gene in ALT tumor cells Was hypermethylated and gene silence. Hypermethylation of The promoter region of p16M4a gene was apparently attenuated and p16M4a gene was de novo activation after2to3days exposure to KBA02and puerh in a concentration dependent manner. DNMT were inhibited by exposure to KBA02and puerh.Conclusions We have demonstrated that KBA02and puerh couLd active demethylation and activation transcription. Hypermethylation of the promoter region of p16Ink4a gene was reversed and activation transcription could be de novo by KBA02and puerh in ALT tumor cells. It may result from down-regulating DNA methyltransferases. To a certain extent, our experiments proved that puerh and Buxaceae as two kinds of adjunctive treatments of some cancers have a very bright future.