The Effects Of DNMT1-siRNA On MiR-148a Expression, CpG Island Methylation Status, And Proliferation Of Pancreatic Cancer BxPC-3Cells

Objective:To explore the effect of DNMT1-siRNA on miR-148a expression, CpG islandmethylation status, and proliferation of pancreatic cancer BxPC-3cells.Methods:Pancreatic cancer BxPC-3cells were cultured in DMEM at37℃and5%CO2incubator and divided into three groups: blank control group, negative control groupand experimental group, which were transfected with empty liposomes, a negativesiRNA and a DNMT1-specific siRNA, respectively. After transfected for48h, theexpression of DNMT1mRNA and protein in the transfected cells was analyzed byreal-time PCR and Western blot, respectively. The expression of miR-148a wasanalyzed by real-time PCR. The CpG island methylation level of miR-148a genepromotor region detected by methylation specific PCR (MSP). Cell proliferation wasmeasured by MTT assay.Results:(1) The relative quantity of DNMT1mRNA in experimental group, negativecontrol group and blank control group were (0.41±0.06),(0.99±0.06) and (1.00±0.00), respectively. The expression of DNMT1mRNA and protein in experimentalgroup was significantly lower than those in the blank control group and the negativecontrol group (P<0.01).(2) The relative quantity of miR-148a in experimental groupwas (3.23±0.17), which was significantly higher than those in negative controlgroup(1.03±0.67) and blank control group(1.00±0.00)(P<0.01).(3) The CpGisland of miR-148a gene promotor region was positive methylation in blank controlgroup and negative control group, while that was negative methylation inexperimental group.(4) The cell proliferation activities in experimental group weresignificantly slower than those in negative control group and blank control group at48h,72h,96h and120h after transfection(P<0.05). The growth inhibition ratiowere12.72%,22.78%,14.44%and18.18%, respectively.Conclusions: Silencing DNMT l gene by RNA interference can specifically suppress theexpression of DNMT1in pancreatic cancer BxPC-3cell, which lead todemethylation for miR-148a gene promoter CpG island and up-regulate theexpression level of miR-148a and inhibit cell proliferation in pancreatic cancerBxPC-3cell. The expression of miR-148a is regulated by the DNA methylation inpancreatic cancer BxPC-3cells. DNMT1and miR-148a can be the effective targetsfor demethylation treatment of pancreatic cancer.