Study On The Mechanism That Silencing SFRP1 Inhibits The Anoikis Of Human Gastric Mucosal Epithelial Cells

ObjectiveAnoikis is a programmed cell death induced upon cell detachment from extracellular matrix or adherent cells.Resistance to anoikis permits gastric cancer cells to survive in systemic or blood circulation and facilitates their metastasis to distant organs.However,the molecular mechanism of anoikis resistance in gastric cancer cells is still unclear.Loss of SFRP1 expression is found in a multitude of cancers including gastric cancer.It is reported that promoter hypermethylation is the reason for it.However,the precise mechanism of SFRP1 in the development of gastric cancer remains to be determined.It is unknown whether SFRP1 plays roles in carcinogenesis of normal gastric epithelial cells.In this study,we inhibited the expression of SFRP1 by RNA interference in gastric mucosal epithelial cells(GES-1)which is anoikis-sensitive.We detected the changes of cell anoikis,anchorage-independent growth,migrative and invasive capacity.Furthermore,we explored the molecular mechanism by which SFRP1 mediates anoikis.Our study will shed new light on the mechanism of loss of SFRP1 expression in the development of gastric cancer.Method1.SFRP1 small interfering RNA(siRNA)and Control siRNA were transfected into GES-1 cells respectively.The SFRP1mRNA and protein expression were detected by Realtime RT-PCR and Western blot.The most effective siRNA was chosen for the following experiment.2.Cells were cultured on poly-HEMA to replicate anoikis environment.3.The effect of SFRPlsiRNA on cell anoikis were conducted by Calcein AM/EthD-1 assay kit and flow cytometry.4.The effect of SFRP1siRNA on the anchorage-independent growth of cells was determined by assaying colony formation in soft agar.5.The effect of SFRP1siRNA on the migration and invasiveness of cells were detected byTranswell chamber.6.Fluorescent immunocytochemistry and western blot were used to detect the localization and expression of β-catenin in Wnt pathway after SFRP1 silencing.7.The c-MycmRNA and CyclinDl mRNA expression were detected by Realtime RT-PCR.Result1.The results of Realtime RT-PCR and Western bolt showed that SFRP1mRNA and protein expression were significantly inhibited insiRNA-891 group compared with control siRNA group(P<0.01 and P<0.05 respectively).2.Calcein AM/EthD-1 fluorescence assay showed that compared with the control group,anoikis cells with EthD-1 fluorescent dye were significantly decreased in siRNA-891 group(P<0.05).Viable cells with Calcein AM fluorescent dye were significantly increased in siRNA-891 group(P<0.05).Flow cytometry results showed that the anoikis rate of siRNA-891 cells was significantly lower than that of control group after 24h of suspension culture(P<0.05).3.The results of soft agar colony experiment showed that the colonies in siRNA-891 group were significantly larger than that of the control group,and the number of colonies was significantly increased(P<0.05).4.The results of Transwell chamber experiment showed that compared with the control group,the migrative and invasive cells in siRNA-891 group were significantly increased respectively(P<0.05).5.The results of immunofluorescence showed increase in nuclear P-catenin accumulation in siRNA-891 group.The results of Western blot showed that expression level of β-catenin protein in siRNA-891 group was significantly higher than that of the control group(P<0.05).6.The results of Realtime RT-PCR showed that expression of c-Myc mRNA and CyclinD1mRNA were significantly increased in siRNA-891 group,compared with the control group(P<0.01).ConclusionInterference of SFRP1 inhibited anoikis sensitivity of GES-1 cells and predisposed it to anoikis resistance.The activation of Wnt pathway maybe one of its mechanism.