The Function And Mechanism Research Of FZD8/Ca²⁺/NFAT In The Drug Resistance Of Philadelphia-positive Acute Lymphoblastic Leukemia

Objective To investigate the role of FZD8/Ca²⁺/NFAT signaling pathway in Philadelphia chromosome-positive（Ph⁺）acute lymphoblastic leukemia drug resistance.Method 1.Collect the newly diagnosed or relapsed Ph⁺ ALL peripheral blood mononuclear cells,detect the expression of FZD8 protein by Western blot,and detect the FZD8 m RNA by PCR;2.Design and synthesis targeting specific interference sequences carrying human FZD8,directionaly connect into the lentiviral vector of LV3.Recombinant lentivirus was generated by co-transfection of three-plasmids mediated by liposome;The SUP-B15 cells were transfected by the lentivirus and screened by Puromycin.As follows,the leukemia cells’ s biological behavior was detected:（1）CCK-8 method to detect changes in leukocyte proliferation ability;（2）Annexin V/7-AAD labeling flow cytometry was used to determine apoptosis rate;（3）flow cytometry was used to detect cell cycle changes of leukemia cells;（4）The transcription levels of FZD8,NFAT,β-catenin and ALDH1 in leukemia cells were detected by real-time PCR.（5）Western blotting was used to detect FZD8,NFAT,CD133 and β-catenin proteins in leukemia cells.3.SUP-B15 cells was seeded in the medium of CSA final concentration of 2.5,5μmol/L respectively,NFAT protein were detected after 24 h,48h and 72 h.SUP-B15 cells was treated with imatinib and CSA alone or combination for 72 h,apoptotic cells ratio was was detected by Annexin V/7-AAD labeling flow cytometry.4.（1）Collecting peripheral blood mononuclear cells of newly diagnosed or relapsed Ph⁺ ALL patients under sterile conditions,cultured alone or co-cultured with bone marrow stromal cells to observe changes in leukemia cell growth status;（2）Bone marrow stromal cells OP9 and SUP-B15 Cells or Ph⁺ ALL primary cells were co-cultured to evaluate the role of FZD8 in the protective effect of bone marrow stromal cells on Ph⁺ ALL cells.Subsequent experimental methods were used: Annexin V/7-AAD labeling flow cytometry to determine apoptosis rate;We used flow cytometry to detect the cell cycle changes of leukemia cells.Fluorescence quantitative PCR was used to detect the transcription levels of FZD8,NFAT,β-catenin in leukemia cells.Western-Blot was used to detect FZD8,NFAT and β-catenin proteins in leukemia cells.Results 1、Peripheral blood mononuclear cells from 6 newly diagnosed and 3 recurrent Ph⁺ ALL patients were collected and FZD8 protein and m RNA expression were successfully detected.However,FZD8 expression was not detected in peripheral blood mononuclear cells of healthy volunteers.2、SUP-B15 cells stably down-regulating FZD8 protein were successfully screened,sh FZD8-2 and sh FZD8-3,respectively,and used in subsequent experiments.The results showed that:（1）The sensitivity of leukemia cells to imatinib was increased after FZD8 was down-regulated（P<0.05）.After down-regulation of FZD8,the proportion of G0/G1 cells in SUP-B15 cells decreased（P<0.05）.The proliferation of leukemia cells was significantly increased after down-regulation of FZD8（P<0.05）.After down-regulation of FZD8 protein expression,the expression of CD133 protein in SUP-B15 cells decreased,and the levels of CD133 and ALDH1 m RNA decreased（P<0.05）.After down-regulation of FZD8 protein expression,The NFAT level of the leukemia cells decreased,the nuclear translocation decreased,and the total protein and nuclear protein β-catenin levels did not change significantly.3、CAS can significantly inhibit NFAT protein expression in clinically applied drug concentrations（2.5,5 μmol/L）;Clinical applied concentration of CAS（2.5,5 μmol / L）has no significant effect on the apoptosis of SUP-B15 cells,while higher concentration of CAS（10 μmol / L）can induce Apoptosis of SUP-B15 cells.Clinically applied CAS concentration（2.5 μmol/L）can significantly increase the sensitivity of SUP-B15 cells to IM.4.The bone marrow stromal cell OP9 has a supporting effect on the primary cells of Ph⁺ ALL,which can significantly reduce the apoptosis of leukemia cells.With the support of OP9,the sensitivity of Ph⁺ ALL primary cells to IM is decreased.The Ph⁺ ALL primary cell XZMC was used for subsequent experiments.After co-culture with bone marrow stromal cells,the levels of FZD8 protein and m RNA in SUP-B15 cells and XZMC cells increased,the total protein levels and nuclear protein levels of NFAT increased,and the total protein levels and nuclear protein levels of β-catenin increased.5、Stable down-regulation of FZD8 expression in SUP-B15 cells partially resisted the protective effect of OP9 cells on SUP-B15 cells;inhibition of Ca²⁺/NFAT signaling pathway by low concentration of cyclosporine partially protected the protective effect of bone marrow stromal cells on Ph⁺ ALL.Conclusion 1、The FZD8/Ca²⁺/NFAT signaling pathway contributes to the survival of Ph⁺ ALL cells.2、Bone marrow stromal cells can mediate the resistance of Ph⁺ ALL cells to IM by activating FZD8/ Ca²⁺/NFAT signaling pathway.