P16^Ink4a Affect Pancreatic Tumor Cell Proliferation Ability Through Inhibiting The Wnt/β-caten In Signaling Pathway

Purpose:1、For the status of a variety of tumor suppressor genes inactivated and oncogenes abnormal activated in pancreatic cancer; Use of5-azacytidine demethylating agent to incubate the pancreatic cancer lines, observed if the5-azacytidine affecte the abnormal methylation of tumor suppressor gene p16Ink4a and cancer-promoting signaling pathway Wnt/β-catenin signaling pathway.2、For the results obtained in the previous step and the status of low expression of p16Ink4a in pancreatic cancer and Wnt/β-catenin signaling pathway activation; observed if the increased expression of p16Ink4a in pancreatic cancer will affect the Wnt/β-catenin signaling pathway activity.Method:Select the human pancreatic cancer cell line Bxpc-3and Miapaca-2cells, incubated in DMEM culture medium,1.we incubate Bxpc-3cells in different concentrations of5-azacytidine (0μmol/L,5μmol/L,10μmol/L) in serum-free DMEM culture medium,for the incubating time is24h,48h,72h, Then we extract the total RNA, p16Ink4a mRNA and the Wnt signaling pathway key signaling molecule β-catenin, c-myc, cyclinDl mRNA expression, total protein was extracted, the protein of p16Ink4a P-catenin, c-myc, cyclinD1were measured by Western-blotting; To determination Bxpc-3cell proliferation effect,we use the CCK8cytotoxicity kit; pancreatic cancer cells Bxpc-3early apoptosis were tested by Flowcytometry.2.Human pancreatic cancer cell line Bxpc-3and Miapaca-2cells, Bxpc-3cells and Miapaca-2cells were transfected with pcDNA3.0which containing the plasmid p16Ink4a gene.The total RNA, total proteins of transfected cells were extracted successfully, the total RNA, total proteins,p16Ink4a β-catenin, c-myc, cyclinDl mRNA and protein expression were also measured, Bxpc-3the ability of cells proliferation. Measured by cytotoxicity kit CCK8.Results:In the influence of5-azacytidine, Bxpc-3cell proliferation was inhibitedunder the different dose and time, Real-time quantitative PCR found at the same point in time with the increase of drug concentration or in the same concentrations as the prolongation, p16Ink4a mRNA and Wnt signaling pathway critical signaling molecule β-catenin, c-myc, cyclinDl mRNA and the protein expression levels of them also were significantly inhibited. β-catenin,cells critical intracellular signaling molecule, has been significantly inhibited by p16lnk4a up-regulation by5-azacytidine treatment, As a result the activity of the Wnt signaling pathway, and the expression of its target gene c-my, cyclinDl expression also was significantly inhibited. Flow cytometry found that as time of drug action and drug concentration is prolonged, Bxpc-3tumor cell early apoptosis was significantly increased, without significant increase in late apoptotic cells.The pcDNA3.0plasmid vector plasmid witch containing p16lnk4a was transfected into two human pancreatic cancer cells, Bxpc-3and Miapaca-2, when transfection is successful, gene expression levels were significantly increased on mRAN levels;This phenomenon was confirmed in the p16protein detection by the way of Western-blotting.while in this experiment also tested the Wnt signaling pathway key signaling molecule β-catenin, c-myc, cyclinDl,The mRNA and protein levels were found:After transfection p16Ink4a into Bxpc-3and Miapaca-2,the β-catenin protein expression was significantly inhibited, whereas in Bxpc-3of c-myc also was inhibited.But cyclinDl mRNA is no significant inhibited,so as to the protein expression in Miapaca-2cells, c-myc protein expression and cyclinDl mRNA and not inhibited significantly, suggesting that there may be other signaling pathways involved in this process, or c-myc and cyclinDl expression may be adjusted by other signaling pathway along with Wnt/β-cantain signal pathwayConclusion:5-azacytidine can increase pl6lnk4a expression, original expression of β-catenin, c-myc and cyclinDl play an inhibitory effect, thereby inhibiting the Wnt/β-catenin signaling pathway, could significantly inhibit pancreatic cancer cell proliferation.The c-myc and cyclinDl is regulated by Wnt/β-catenin signaling pathway in tumor cells as an effector molecules has also been inhibited, but its function is regulated by multiple signaling pathways。5-azacytidine can significantly inhibit the proliferation of pancreatic cancer cells, its mechanism for the inhibition of Wnt signaling pathway in pancreatic cancer cell though the inhibiting of p16Ink4a expression, it aslo influence the activity of c-myc and cyclinDl expression。we transfected p16Ink4a genes into Bxpc-3and Miapaca-2tumor cells, we found that p16Ink4a genes enhancements can affect Wnt/β-catenin signaling pathway in pancreatic cancer cells。but the c-myc and cyclinD1,as two kinds of downstream factor of Wnt/β-catenin signaling pathway effector molecules is no significant effect; The specific molecular mechanisms need further study.