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AIM:To elucidate the role of dickkopf3(Dkk3)in human pancreatic cancer cell growth.METHODS:Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction(realtime RTPCR),Western blotting and immunofluorescence.Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction(MSP)and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2’deoxycytidine(5azadC)treatment.The effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96?AQueous One Solution Cell Proliferation Assay(MTS)after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells.The expression ofβcatenin,phosphorylated extracellular signalregulated protein kinases(pERK)and extracellular signalregulated protein kinases(ERK)was also examined by realtime RTPCR and Western blotting after upregulating Dkk3 expression in human pancreatic cancer cells.RESULTS:The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested.The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines,which showed reduced Dkk3 expression.These two cell lines,which initially had a methylated Dkk3 promoter,showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent,5azadC,treatment(P<0.05 or P<0.01).When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells,the ability of cells to proliferate decreased(P<0.01),and the expression ofβcatenin and pERK was downregulated(P<0.01).Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmidtransfected cells.CONCLUSION:Our findings,for the first time,implicate Dkk3 as a tumor suppressor in human pancreatic cancer,through the downregulation ofβcatenin expression via the ERKmediated pathway.
AIM: To elucidate the role of dickkopf3 (Dkk3) in human pancreatic cancer cell growth. METHODS: Dkk3 mRNA and protein expression in human pancreatic cancer cell lines were detected by realtime reverse transcription polymerase chain reaction (realtime RTPCR), Western blotting and immunofluorescence. Methylation of the Dkk3 promoter sequence was examined by methylationspecific polymerase chain reaction (MSP) and Dkk3 mRNA expression was determined by realtime RTPCR after 5aza2 ’deoxycytidine (5azadC) treatment. These effects of Dkk3 on cancer cell proliferation and in vitro sensitivity to gemcitabine were investigated by CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) after transfecting the Dkk3 expression plasmid into human pancreatic cancer cells. The expression of βcatenin, phosphorylated extracellular signalregulated protein kinases (pERK) and extracellular signalregulated protein kinases (ERK) was also examined by realtime RTPCR and Western blotting after upregulation Dkk3 expression in hu man pancreatic cancer cells .RESULTS: The results show that the expression levels of both Dkk3 mRNA and protein were low in all pancreatic cancer cell lines tested. The Dkk3 promoter sequence was methylated in the MIA PaCa2 and AsPC1 cell lines, which showed reduced Dkk3 expression These two cell lines, which initially had a methylated Dkk3 promoter, showed increased Dkk3 mRNA expression that was dependent upon the dosage and timing of the DNA demethylating agent, 5azadC, treatment (P <0.05 or P <0.01) .When Dkk3 expression was upregulated following the transfection of a Dkk3 expression plasmid into MIA PaCa2 cells, the ability of cells to proliferate decreased (P <0.01), and the expression of β catenin and pERK were downregulated (P <0.01). Sensitivity to gemcitabine was enhanced in Dkk3 expression plasmid transfected cells. CONCLUSION: Our findings, for the first time, implicate Dkk3 as a tumor suppressor in human pancreatic cancer, through the downregulation of β catenin expression via the ERKmediated pathway.