| ObjectiveTo explore the mechanism of miR-711 inhibited gastric cancer cell stem and epithelial-mesenchymal transformation of(EMT)through PI3K/AKT/Snail pathway,so as to provide theoretical basis for targeted therapy of gastric cancer.Methods1.Taking SGC-7901 cells as the research object,CD44+SGC-7901 cells were isolated by magnetic beads separation.2.MiR-711 mimics and inhibitor were transfected into SGC-7901 cell line and CD44+SGC-7901 cell line by liposome(3000)transfection method.3.Changes in mRNA expression and expression of stem cell-specific signa ling molecules(CD44,Oct4,Nanog and CD133)and molecules related to the EMT(E-cadherin,Vimentin,MT1-MMP,Snail)were detected by qRT-PCR and WB.4.Cell proliferation capacity in each group was assessed by the CCK8.5.Application of scratch detection and transfer(Transwell)detection to det ermine the transfer and erosion ability of relative somatic cells.6.We took the dry ball-forming test to analyze the dry ball-forming capac ity of cells in each group.7.The expression levels of Snail and E-cadherin mRNA were detected by LY294002,qRT-PCR assay with PI3K specific inhibitor in each group.Results1.CD44~+SGC-7901 cells have the features of stem cells in gastric cancer1)qRT-PCR and Western blotting(WB)data showed that the SFM grou p(SGC-7901 somatic cells shaped in serum-free protein culture medium)Oct4,Nanog and CD133 stem cell markers were higher than the control Group(SG C-7901 cells shaped in all normal culture medium).The corresponding CD44+group(CD44~+SGC-7901 cell group sorted by magnetic bead sorting)was also significantly higher than that of SFM(P<0.05).2)The results of CCK8 experiment showed that 48 hours later,the numb er of cells in CD44+group was significantly higher than that in SFM group(P<0.05).3)Scratch test showed that compared with the SFM and the control grou p,the scratch width of CD44~+SGC-7901 was 10.23±0.97<18.16±0.68<21.94±2.21.The results showed that after the magnetic bead screening,CD44 SGC-7901 somatic cell transfer ability is stronger(P<0.05).4)Transwell analysis showed that the number of somatic cells in the CD44~+SGC-7901 group was 1.92 times higher than that in the SFM group(P<0.05).5)The dry method granulation experiment showed that in the low adhesio n cell culture dish with special cell growth factors,the selected CD44~+SGC-7901 somatic cells can produce more than one hundred somatic cells(per hundr ed individual cells P has more somatic cells than SFM group(P<0.05).2.miR-711 inhibits the dryness of CD44~+SGC-7901 somatic cells1)The results of qRT-PCR showed that in CD44+group and SGC-7901group,the mRNA level of miR-711 in the cell line transfected with miR-711mimics was up-regulated compared with that in the control group,and the m R NA level of miR-711 in the cell line transfected with inhibitor was down-regul ated compared with that in the control group(P<0.05),and the difference was s tatistically significant.2)The levels of mRNA、protein in the CSC tags of(CD133,Oct4,Nano g)in GC cells were tested by qRT-PCR/WB.The results showed,miR-711 mim ics group had lower content than the control group.On the contrary,the transf ected miR-711 inhibitor cell line was increased relative to the control group an d the difference was obvious(P<0.05).3)Cell migration(Transwell)assay showed that in CD44+group and SG C-7901 group,the number of migrating cells transfected with miR-711mimics c ell line was down-regulated compared with that in control group,while the nu mber of migrating cells transfected with miR-711inhibitor cell line was higher t han that in control group,and the difference was statistically significant.(P<0.05).4)Scratch healing experiment showed that the average scratch width of tr ansfected miR-711mimics cell line in CD44+group and SGC-7901 group was wider than that in control group,and the average scratch width of transfected miR-711inhibitor cell line was narrower than that in control group(P<0.05).5)Dry spheroidization test showed that the number of globules in transfec ted miR-711mimics cells in CD44+group and SGC-7901 group was significant ly lower than that in control group,while that in miR-711 inhibitor cells was significantly higher than that in control group(P<0.05).3.miR-711 inhibits the epithelial cytoplasmic transition in CD44~+SGC-7901 somatic cells(EMT)1)Determination of the EMT signal content(Vimentin;MT1-MMP;E-cad herin):QRT-PCR/WB were used to test the mRNA/protein levels.The results s howed:CD44~+SGC-7901,all of the levels of protein or mRNA、E-cadherin tr ansfected miR-711 significantly mimicsked the cell line compared to the contro l group,and Vimentin and MT1-MMP decreased significantly compared to the control group.Transfection with the miR-711 inhibitor cell line was reversed(P<0.05).4.miR-711 exerts inhibitory effect by regulating PI3K/Akt/Snail signal path way1)The results of Western blotting(WB)showed that the P-PDK1 and P-AKT of transfected miR-711 mimics cells in CD44+group and SGC-7901 gro up were significantly lower than those in control group,while the P-PDK1 and P-AKT of miR-711 inhibitor cells were significantly higher than those in cont rol group(P<0.05).2)The results of qRT-PCR detection showed that in CD44+group and S GC-7901 group,the Snail of transfected miR-711 mimics cells was significantl y lower than that of control group,and the Snail of transfected miR-711 inhibi tor cells was significantly higher than that of control group(P<0.05).3)The results of qRT-PCR detection showed that in CD44+group and S GC-7901 group,the level of Snail mRNA after treatment with PI3K specific i nhibitor LY294002 was significantly lower than that after treatment with dimet hyl sulfoxide,and the level of E-cadherin mRNA after treatment with PI3K sp ecific inhibitor LY294002 was significantly higher than that after treatment wit h dimethyl sulfoxide(P<0.01).Conclusions1.CD44~+SGC-7901 somatic cells have the beauty characteristics of rectal cancer stem cells.2.MiR-711 may inhibit the occurrence of(EMT)in dry gastric cancer cel ls and epithelial-mesenchymal transformation of CD44+SGC-7901 gastric cance r cells through PI3K/AKT/Snail pathway. |
PDF Full Text Request