The Role Of Na +-k +-ATPase In Bufalin Anti-bladder Tumor And Its Related Molecular Mechanism

Objective: To study the role of Na +-K +-ATPase in Bufalin anti-bladder tumor and its related molecular mechanismMethods: 1.Join bufalin to bladder cancer T24 cells when it growth stable and than observation bufalin on human bladder cancer T24 cell proliferation bufalin on cell morphology.2.MTT assay was used to detect the inhibitory effect of bufalin on the proliferation of bladder cancer T24 cells.3.The expression of Na +-K +-ATPase ?1,?2,?3 subunit in bladder cancer T24 cells was detected by fluorescence quantitative PCR in the presence of bufalin 48 h IC50 and screening out the key Na +-K +-ATPase subunits.4.Construction of bladder cancer T24 Na +-K +-ATPase key subunit silencing lentiviral vector,and transfected T24 cells.The optimal silencing strain was screened by fluorescence quantitative PCR.The morphological changes of human bladder cancer T24 cells were observed in cell morphology after silencing Na +-K +-ATPase key subunit.5.FCM detection of Na +-K +-ATPase key subunit silencing after bufalin effect on T24 cell apoptosis rate changes;Results:1.Bufalin had a significant inhibitory effect on T24 cells.T24 cells were added with a large number of apoptotic cells,and the number of cells was reduced,the cell volume became smaller,rounded,shrinkage,shedding,suspended in the medium;2.MTT assay was used to detect the inhibitory rate of Bufulanin on bladder cancer T24 cells.The results showed that toadfen had a significant inhibitory effect on bladder cancer T24 cells.With the increase of bufalin concentration the inhibition rate increases,the IC50 was 18.6nmol / L at 48 h.3.The expression of Na +-K +-ATPase ?1,?2,?3 subunit in bladder cancer T24 cells was detected by fluorescence quantitative PCR..The expression of ? subunit was 1 in blank control group,and the results were as follows: The relative expression of Na +-K +-ATPase ?1 gene in T24 cells was(1.86 ± 0.12);The relative expression level of Na +-K +-ATPase ?2 gene was(0.94 ± 0.18);The relative expression level of Na +-K +-ATPase ?3 gene was(14.68±0.56).The expression of Na +-K +-ATPase ?3 was significantly increased after the action of bufalin,and the increase of ?1 and ?2 subunits was not significant(P <0.05),So the choice of Na +-K +-ATPase ?3 for the virus silence point line of the next step.4.Construction of bladder cancer T24 Na +-K +-ATPase key subunit silencing lentiviral vector and use the fluorescence quantitative PCR to screening the best silencing effect of the virus strain,the result show that the expression levels of each Na +-K +-ATPase ?3 subunit RNA in the blank control group,sh RNA1,sh RNA2,sh RNA3 and negative virus were(1.03±0.02)?(0.04±0.01)?(0.85±0.34)?(0.81±0.26)?(0.90±0.02),P <0.05,the difference was statistically significant.So we choose the sh RNA1 as a positive interference sequence for follow-up study.5.After transfection of T24 cells,the effect of bufalin on the morphology of T24 cells was significantly decreased,and the number and morphological changes of T24 cells were not changed significantly compared with the control group.6.The apoptosis rate of T24 cells in the negative virus group was(68.42 ± 1.74)%,and the experimental group of the Na +-K +-ATPase ?3 subgroup was added to the T24 cells The apoptotic rate was(28.36±0.42)%,the difference was statistically significant(P <0.05).Conclusion: Bufalin has a significant inhibitory effect on bladder cancer T24 cells and inhibits the growth of bladder cancer cells;The ability of Bufalin to inhibit the growth of bladder cancer T24 is related to the expression of Na +-K +-ATPase ?3 in bladder cancer T24 cells;When T24 cells Na +-K +-ATPase ?3 were disturbed,bufalin inhibited the growth of bladder cancer cells,Na +-K +-ATPase ?3 may be an important target for the growth of bladder cancer cells.