Effect Of Wnt5a On Centrosome Separation And Inhibiting Proliferation In U937Cells

Objective:Wnt5a is a member of Wnts family which are secreted glycoprotein signalingmolecules, and playes an important role in mediating vital biological processes, such asembryogenesis and tumorigenesis. Wnt5a can ignite different signaling pathway and playdifferent roles. For example, Wnt5a playes a tumour-promoting or tumour-suppressingrole in tumorigenesis. Nowadays, some studies have reported that Wnt5a atumou-suppressing factor was downregulated in some kinds of leukaemia, and recoveredin complete remission leukemic cases. In our previous study, we have demonstrated thatexogenous Wnt5a can decrease the expression of β-catenin in U937cells, block theprocess of mitosis and inhibit the cells malignant proliferation. However, the mechanismis still obscure. In this study, we apply the following methods Western blot,immunofluorescence, immunoprecipitation and Si-RNA etc. to explore the mechanism.We hope to verify whether the Wnt5a downregulates the β-catenin expression via theRor2/JNK/GSK-3β pathway and decreases the expression of its target genes, and whetherthe phosphorylated and decreased β-catenin can inhibit the centrosome separation andblock the mitosis of U937cells. The study explore the mechanism of Wnt5a as atumou-suppressing factor and is beneficial to the therapy of leukemia.Methods:1. The effect of Wnt5a on the β-catenin expression and the cell proliferation inmyeloid leukemic cells1.1RT-PCR and MSP (Special PCR) were used to detect the expression of Wnt5aand its promotor methylation in myeloid leukemic cases. Immunocytochemistry andWestern blot were used to detect the expression of Wnt5a and β-catenin in U937cells andmyeloid leukemic cases.1.2Western blot was used to detect the expression of β-catenin and its target gene(Cyclin D1, C-myc) after the U937cells were treated with Wnt5a.1.3The transcriptional activation of β-catenin was detected by the dual luciferasereporter system after the U937cells were treated with Wnt5a.1.4The proliferation activity was detected by the MTT and flow cytometry after theU937cells were treated with Wnt5a.2. The mechanism of that Wnt5a reduces β-catenin expression through Wnt5a/Ror2pathway in U937cells.2.1The expression of Ror2and LRP5receptor was detected byimmunocytochemistry in acute myeloid leukemic and health cases.2.2The immunofluorescence was used detect the location of Wnt5a, Ror2and LRP5.The immunoprecipitation was used detect the combination of Wnt5a with Ror2and LRP5.The expression of Ror2and LRP5was detected by Western blot.2.3The expression of JNK-p, GSK-3β-p, GSK-3β, β-catenin-p and β-catenin wasdetected by Western blot under some kinds of conditions(Ror2Ab, LiCl and SP600125)after the U937cells were treated with Wnt5a.3. The effect of Wnt5a on centrosome separation and proliferation in U937cellsthrough Ror2pathway3.1Western blot was used to detect the expression of β-catenin at centrosome undersome kinds of conditions(such as Ror2Ab, Ad-Si-β-catenin) after the U937cells weretreated with Wnt5a.3.2The immunofluorescence was used to detect the effect of β-catenin oncentrosome separation3.3The immunofluorescence was used to detect the location of GSK-3β andβ-catenin. The immunoprecipitation was used detect the combination of GSK-3β withβ-catenin at the centrosome. The phosphorylated β-catenin at the centrosome was detectedby Western blot under some kinds of conditions Ror2-Ab, LiCl and SP600125after theU937cells were treated with Wnt5a.3.4The immunofluorescence was used detect the effect of phosphorylated β-cateninon centrosome separation. Results:1. The expression of Wnt5a was downregalated owing to the methylation in myeloidleukemic cases. And β-catenin expression was upregalated owing to the low expression ofWnt5a in U937cells.2. The transcriptional activation of β-catenin was inhibited owing to the lowexpression of β-catenin regulated by exogenous Wnt5a in U937cells, which candownregalate the expression of cyclin D1and C-myc genes.3. The process of mitosis was blocked, and the manignant proliferation was inhibitedafter the U937cells were treated with exogenous Wnt5a.4. The expression of Ror2receptor was higher than the expression of LRP5receptorin healthy cases compared with the acute myeloid leukemic cases.5. Wnt5a can bind to the Ror2receptor prior to the LRP5receptor, furthermore canupregulate the expression of Ror2receptor in U937cells.6. The expression of β-catenin was downregulated by the combination of Wnt5a toRor2receptor in U937cells.7. The expression of β-catenin at centrosome was downregulated through theJNK/GSK-3β pathway after the combination of Wnt5a to Ror2receptor in U937cells,which can block the separation of centrosome in mitosis.8. The centrosome separation was inhibited by the increased phosphorylatedβ-catenin regulated by Wnt5a/Ror2/JNK/GSK-3β pathway in U937cells.Conclusion:Wnt5a can influence the function of β-catenin, the key molecule in Wnt canonicalsignaling pathway, through the Ror2/JNK/GSK-3β pathway in the myeloid leukemic U937cells, acting as a tumor suppressing factor of leukaemia. Wnt5a can reduce the expressionof β-catenin in U937cells, which results in that Wnt5a can antagonize Wnt canonicalsignaling pathway, downregalate the expression of Cyclin D1and C-myc genes and inhibitthe malignant proliferation of U937cells, and that impact the centrosome separation,spindle formation and block the process of mitosis in U937cells.