| Objective Study on the effects of silencing neuropilin1 on migration,invasion and Epithelial-Mesenchymal Transition(EMT)in gastric cancer HGC-27 cells.Methods During this study,using TGF-β1 to induce EMT model in gastric cancer HGC-27 cells.Then HGC-27 cells were stably transfected with si RNA targeting NRP1.Wound healing and transwell assays were used to measure cell migration and invasion.NRP1 and EMT markers(E-cadherin,vimentin,snail)expression levels were measured using quantitative real-time reverse transcription-polymerase chain reaction(q RT-PCR)and Western blotting assay.Results 1 TGF-β1 induced EMT model of gastric cancer HGC-27 cell: TGF-β1 5 ng/mL is used to stimulation group cell.Morphology of oval-like to fusiform,spindle-shape change and the cell gap increase can observed.Scratches showed that the area of scratches in the stimulation group was significantly increased,compared to the blank group(P < 0.05).Transwell results showed that the number of cells in the stimulation group penetrating the cells was significantly higher than that in the blank group(P < 0.05).qRTPCR results showed that after TGF-β1 stimulated cells,the relative m RNA expression of E-cadherin was significantly reduced,and the relative m RNA expression of Vimentin,Snail and NRP1 was significantly increased(P < 0.05).Western blot results showed that the E-cadherin protein in the stimulation group was significantly decreased,and the expression of Vimentin,Snail and NRP1 protein was significantly increased(P < 0.05).Verify successful establishment of EMT model.2 Silence of NRP1 in gastric cancer HGC-27 cell line: To verify transfection efficiency.Compared with the CON(blank control group,transfected with PBS)and NC(Non-sequence group,transfected with NC-si RNA)groups,q RT-PCR and Western blot results showed that,the relative expression levels of NRP1 m RNA and protein in gastric cancer HGC-27 cells in the si NRP1 transfected group(transfected group,transfected with NRP1-si RNA)were significantly reduced(0.47 fold)(P < 0.05).Verify that the transfection silence effect is satisfactory.3 Gene silencing NRP1 can inhibit the migration and invasion of gastric cancer HGC-27 cells: in the transfection experiment,the wound healing area of the si NRP1 group was significantly reduced compared with the CON group and the NC group at 24 and 48 h after scratching(P < 0.05).Transwell experiments showed that the number of migrated and invaded cells in the si NRP1 group was decreased,compared with the CON and NC groups(P < 0.05).4 Silencing NRP1 can inhibit invasion,migration and reverse EMT process after TGF-β1-induced EMT in HGC-27 cells: After 24 and 48 hours of scratching,the wound healing of TGF-β1 + si NRP1 group was less than that of other groups(P < 0.05);Transwell experiment showed that the number of migrating and invasive cells in TGF-β1 + si NRP1 group was significantly reduced compared with other groups(P < 0.05).After TGF-β1 induced EMT,transfection of si NRP1 to silence NRP1.Experiments were design into four groups:(1)Blank group(without treatment)(2)TGF-β1 + CON group(cells treated with TGF-β1 and transfected with the PBS)(3)TGF-β1 + NC group(cells treated with TGF-β1 and transfected with the negative control si RNA)(4)TGF-β1 + si NRP1 group(cells treated with TGF-β1 and transfected with NRP1-si RNA).Comparison between each groups,q RT-PCR and Western blot results showed that the expression of E-cadherin in the TGF-β1 + si NRP1 group was significantly increased,and the expression levels of Vimentin and Snail were significantly down-regulated(P < 0.05)Conclusion Under the condition of TGF-β1 induced establishment of gastric cancer EMT,silencing NRP1 can inhibit cell migration,invasion and reverse the TGF-β1 induced EMT process of gastric cancer.Figure 4;Table 2;Reference 147... |
