Blockade Of Wnt1 Signaling Pathway By RNAi Technology Can Inhibite Proliferation Of Human Neuroblastoma SH-SY5Y Cells

Backgrounds and purpose:Neuroblastoma(NB),derived from peripheral sympathetic nervous system, which originates from the neural crest,is the most common malignant tumor in children.Our previous study showed that neuroblastoma was intimately associated with the abrerrant embryonal development.The Wnt signaling pathway takes important roles in embryonic development,including cell proliferation,cell differentiation and cell migration.Defects in this pathway have been implicated in the pathogenesis of various types of tumors in human beings.The canonical Wnt signaling consists of an intracellular cascade that involves Fz,Dishevelled(Dvl), glycogen synthase kinase-3β(GSK-3β),β-catenin and T Cell Factor (TCF)/Lymphocyte Enhancer Binding Factor(LEF),etc..In the presence of Wnt signals,Wnt ligands binding to their Fz receptors leads to phosphorylation and increased activity of Dvl.Phosphorylated Dvl inhibits the phosphorylating activity of GSK-3β.The regulation of GSK-3βis mediated through casein kinase-1(CK-1), which phosphorylates Dvl.This,in turn,prevents GSK-3βfrom phosphorylating its substrates,critically decreasing the binding affinities of the negative regulators Axin and APC(adenomatous polyposis coli) forβ-catenin.Unphosphorylatedβ-catenin escapes recognition by aβ-transducing repeat-containing protein,a component of an E3 ubiquitin ligase.Asβ-catenin accumulates,it translocates into the nucleus,where it binds to TCF/LEF transcription factors to form a complex that activates transcription of downstream target genes,such as cyclinD1,c-myc,etc.,and then regulates cell growth,cell development and cell differentiation.In the absence of Wnt signals,free cytosolicβ-catenin is incorporated into a complex consisting of Axin,APC gene product,and GSK-3β.Conjunctional phosphorylation of Axin,APC, andβ-catenin by GSK-3βdesignatesβ-catenin for the ubiquitin pathway,where ubiquitination ofβ-catenin targets it for degradation by proteasomes.Acumulation ofβ-catenin in nuclei is a key mediator to induce tumorigenesis.In the recent study,we find that Wnt proteins and its subordinate signaling take part in the neural crest cell development and differentiation.Within the Wnt proteins,overexpression of Wnt1 induced neural crest cell increasing.Inhibiting Wnt1 induced neural crest derivate to be deficient and nervous precursor cell to decrease.Wnt1 can regulate neural crest cell,which induced by nerve growth factor(NGF),differentiation.Therefor,in our experiment,the expression of Wnt1 in human neuroblastoma SH-SY5Y cells was detected.And then siRNA targeting Wnt1 was used to observe inhibitory efect of Wnt1 signaling pathway on the growth of human neuroblastoma SH-SY5Y cells,and to verify the relationship between the aberrant Wnt1 signaling pathway and neuroblastomas.Materials and Methods:Human neuroblastoma SH-SY5Y cells were purchased from cell center of Peking Union Medical University.Anti-Wnt1 antibody was purchased from Peking Aviva biotechnology limitted company,and anti-β-catenin antibody from Sant Cruz. SiRNA was made by Shanghai Jima biotechnology company.SH-SY5Y cells were divided into four groups,including negative control,vector,nonsense-siRNA and Wnt1-siRNA group.PCR,Western blot and immunofluorescence technology were used to test the expression of Wnt1 in human neuroblastoma SH-SY5Y cells on three levels of gene,protein and location.RNAi technology was used to observe its inhibitory effect on the growth of the cells.SiRNA targeting Wnt1 was transfected into SH-SY5Y cells.The protein expression of Wnt1 andβ-catenin were detected by Western blotting after transfection.The quantity and the morphologic changes were observed by light microscope.The growth rate of SH-SY5Y cells after RNAi transfection was studied by MTT assay.Results:Wnt1 expressed well in human neuroblastoma SH-SY5Y cells.And the cells were transfected with siRNA targeting Wnt1 mediated by Lipofectamine in vitro. The transfection was successful.The proteins expression of Wnt1 andβ-catenin were decreased after transfection with siRNA.The numbers and the synapses of the cells were decreased,accompanying with lots of floating and dead cells under the light microscope.SH-SY5Y cells transfected with siRNA targeting Wnt1 showed lowering proliferation activity. Conclusion:1.Wnt1 expressed well in human neuroblastoma SH-SY5Y cells.2.Wnt1 signaling pathway may play a very important role in the proliferation of Human neuroblastoma SH-SY5Y cells.3.Wnt1 can be the candidate gene for gene therapy of malignant neuroblastomas.