| Part 1:Using Gene Chip to analysis the expression of genes after CtBP2 silence in ECA109 cellsObjective:To better understand the function of C-terminal binding protein 2(CtBP2)in Esophageal squamous carcinoma cells EC A109,we synthesized CtBP2 small interfering RNA(siRNA)and used Gene Chip to find some useful target genes.Methods:(1)100nM siRNA(including CtBP2 siRNA and negative control NC siRNA)were mixed with 6μL transfection regent,then added into the cultured ECA109 cells;(2)72h later,we extracted the RNA in both groups to analysis by using Gene Chip;(3)The total protein in both groups was extracted to do Western blot.Results:(1)We found 166 genes,43 genes upregulated after treatment of CtBP siRNA,and 123 genes downregulated.In those upregulated genes,we found fibroblast growth factor 2(FGF2);(2)The result of Western blot showed that the FGF2 was downregulated after treatment of CtBP2 siRNA(*P<0.05).Conclusion:CtBP2 adjusted the expression of FGF2,and FGF2 may be an important factor in ECA109 cells.Part 2:The effects of FGF2 silence in the proliferation,migration and invasion of ECA109 cellsObjective:We acquired FGF2 silence Lentivirus vectors in vitro using RNA interference(RNAi),and screened the cells which stably expressed low level FGF2 by using puromycin,and to illustrate the the effects of FGF2 silence in the proliferation,migration and invasion of EC A109 cells.Methods:(1)We got the RNAi sequence(5,-CGAACTGGGCAGTATAAAC-3 ’)to construct lentivirus;(2)1×105 cells were seeded into 6-well plate,the lentivirus were added in to ECA109 cells at MOI=5,then screened by puromycin;(3)2×103 cells were seeded into 96 well plate,and the CCK-8 agent was added into 96 well plate at 1,2,3,4,5d,then the OD450 value was calculated by microplate reader;(4)1×105 cells was seeded into 6 well plate,24h later,the cell cycle were got by flow cytometry;(5)5×104 cells were seeded into 24 well plate,and 24h later,50μM EdU were used to detected the cells at S-phase;(6)2×10 cells were seeded into the small wells in 35mm-μ Dish,and the insert was disposed when the cells confluence was 90%,the images were taken at 24h and 48h;(7)2×104 cells were seeded into upper chamber which precoated by matrigel,16h later,the crystal violet was used to label cells;(8)1×105 cells were added in to 6 well plate,48h later,the total protein were acquired and the Western blot was used to detect the expression of p-AKT 和 p-mTOR.Results:(1)The green fluorescence could be detect in LV-FGF2-RNAi、LV-scr-RNAi groups,but not in Wide type(WT)group;(2)The cells viability in LV-FGF2-RNAi was decreased at the 2d and continued to the 5d(*P<0.05);(3)The cells at S-phase in LV-FGF2-RNAi group(21.38 ±0.83%)is lower than others(WT:31.22±1.02%,LV-scr-RNAi:31.30±1.20%)(***P<0.001);(4)The EdU positive cells(29.22± 1.83%)in LV-FGF2-RNAi group was also lower than other groups(WT:46.32±1.62%,LV-scr-RNAi:48.95±2.81%)(***P<0.001);(5)The migration distance in LV-FGF2-RNAi group was less than WT and LV-scr-RNAi groups(***P<0.001);(6)The numbers which passed the upper chamber in LV-FGF2-RNAi group was less than other groups(WT:132.89±9.20,LV-scr-RANi:122.70±10.27,LV-FGF2-RNAi:83.87± 21.96)(**P<0.01);(7)The protein expression of p-AKTand p-mTOR was decreased after FGF2 RNAi(***P<0.001).Conclusions:Using RNAi method to decrease the intrinsic expression of FGF2 could inhibit the proliferation,migration and invasion of ECA109 cells.Part 3:Exogenous FGF2 promotes the proliferation,migration and invasion of ECA109 cellsObjective:The extrinsic FGF2 was added into medium and to evaluate the effects of exogenous FGF2 in ECA109 cells.Methods:(1)2×103 cells were seeded into 96 well plate with 2ng/mL、10 ng/mL、20 ng/mL、50 ng/mL、100 ng/mL FGF2 respectively,then CCK-8 regent was added into 96 well plate at 1,2,3,4,5d,then the OD450 value was calculated by microplate reader;(2)1×105 cells was seeded into 6 well plate and then 20ng/mL FGF2 combined with/without 100 u M LY294002 was added into medium,24h later,the cell cycle were got by flow cytometry;(3)5×104 cells were seeded into 24 well plate and then 20ng/mL FGF2 combined with/without 100 u M LY294002 was added into medium,and 24h later,50 u M EdU were used to detected the cells at S-phase;(4)2×104 cells were seeded into the small wells in 35mm-μ Dish;when the cells confluence was 90%the insert was discarded and then 20ng/mL FGF2 combined with/without 100μM LY294002 was added into medium,all the images were taken at 24h and 48h;(5)2×104 cells were seeded into upper chamber which precoated by matrigel,then 20ng/mL FGF2 combined with/without 100μM LY294002 were added into lower chamber;12h later,the crystal violet was used to label cells;(6)1×105 cells were added in to 6 well plate,then 20ng/mL FGF2 combined with/without 100μM LY294002 were added into the medium;48h later,the total protein were acquired and the Western blot was used to detect the expression of p-AKT 和p-mTOR.Results:(1)The viability of cells was higher than controls after treatment by FGF2 at the 4d;but the viability in varied concentrations didn’t show any difference;the 100μM LY294002 inhibited the proliferated effect which induced by 2ng/mL FGF2;(2)The cells at S-phase in FGF2 group is higher than others,but the S-phase cells decreased after treatment of LY294002(0ng:37.18±0.54%,2ng:43.52±0.59%,2ng±LY294002:29.70±0.28%,LY294002:27.25±0.55%)(***P<0.001);(4)The EdU positive cells(55.07±1.89%)in FGF2 group was also higher than other groups(**P<0.001),but decreased after treatment of LY294002(35.16±2.66%);(5)The migration distance in FGF2 group was more than other groups,but but decreased after treatment of LY294002;(6)The numbers which passed the upper chamber in FGF2 group(67.33±6.33)was more than other groups(***P<0.001);but decreased after treatment of LY294002(26.33 ± 1.81);(7)The protein expression of p-AKTand p-mTOR was increased after treatment of FGF2,but decreased after used of LY294002.Conclusions:The exogenous FGF2 can promote the proliferation,migration and invasion of ECA109 cells;and the effects of FGF2 in ECA109 cells was associated with the PI3K/AKT signaling pathway. |
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