Study On The Effect Of S100A8/A9 Protein On Migration And Proliferation Of Neuroblastoma And Its Mechanism Through WNT/?-catenin Pathway

Objective: Neuroblastoma(NB)is an extracranial malignancy that occurs most frequently in children and accounts for about 7% and 15% of the morbidity and mortality of childhood malignancy.S100A8 and S100A9 are important members of the calcium-binding protein S100 superfamily,which usually exist in the form of dimers in cells and can play an important role as inflammatory factors in the regulation of inflammatory response.Recent studies have shown that S100A8/A9 expression is abnormal in a variety of tumor tissues,which is closely related to the occurrence and development of tumors,but the relationship between S100A8/A9 and neuroblastoma has not been clearly and deeply reported.Therefore,this study aims to observe whether S100A8/A9 can affect the proliferation and migration of neuroblastoma through the WNT/ ?-catenin pathway on the basis of the previous study and in vitro tumor formation experiments,and explore its molecular mechanism,so as to provide theoretical research basis for the occurrence and development of NB.Methods:(1)Bioinformatics analysis of S100A8/S100A9 expression in neuroblastoma.(2)Recombinant plasmids constructed by the research group with green fluorescent markers [overexpression group: S100A8-SBI-piggbac(SBI-S100A8)and S100A9-SBI-piggbac(SBI-S100A9);low-expression group: shRNA-S100A8(shS100A8 or shA8)and shRNA-S100A9(shS100A9 or shA9)] transfected with NB cell line SH-SY5 Y cells.After purinamycin screening,stable cell lines with overexpression of S100A8/A9 and underexpression of S100A8/A9 were constructed.(3)The proliferation of NB was observed in plate cloning experiment;NB migration and invasion were observed by scratch,Transwell and invasion experiments,and tumor growth was observed by subcutaneous tumor formation in nude mice.(4)The adenovirus AdGFP,AdRFP,Adsi?-catenin and Ad?-catenin were used to treat NB cell lines and their stable translocation.(5)S100A8,S100A9 and ?-catenin proteins were detected by Western blotting.Results:(1)Bioinformatics analysis showed that S100A8/A9 protein was highly expressed in neuroblastoma.(2)The molecular experiments and fluorescence expression proved that the stable strain construction was successful.(3)Results of Transwell migration and invasion experiment,scratch and plate cloning experiment: compared with the control group,the neuroblastoma with overexpression of S100A8,S100A9 and overexpression of ?-catenin protein had increased proliferation and migration capacity(P<0.05),while the neuroblastoma with low expression had decreased proliferation and migration capacity(P<0.05).(4)Results of Transwell migration and invasion experiment,scratch experiment and plate cloning experiment: when S100A8/A9 expression was high and the expression of ?-catenin protein was low,the proliferation and migration ability of neuroblastoma decreased compared with the S100A8/A9 overexpression group(P<0.05).(5)Subcutaneous tumorigenesis experiments in nude mice showed that the growth of neuroblastoma in the group with high expression of S100A8,S100A9 and the group with high expression of ?-catenin protein increased significantly,which was different from the group with low expression and the control group(P<0.05).Subcutaneous tumorigenesis experiments in nude mice also showed that: when S100A8/A9 expression was high and the expression of ?-catenin protein was low,compared with the S100A8/A9 expression group,the growth of neuroblastoma was inhibited to some extent.Conclusion: The above results showed that the high expression of S100A8,S100A9 and ?-catenin protein can induce the growth,migration and invasion of SH-SY5 Y in neuroblastoma cells,while the low expression can lead to inhibition.S100A8 / A9 promotes the proliferation and migration of neuroblastoma by affecting the hyperexpression of ?-catenin,and S100A8 / A9 activates ?-catenin by activating the classical pathway of WNT to regulate neuroblastoma by positive feedback.This indicated that S100A8 / A9 affected the progress of neuroblastoma through the WNT / ?-catenin pathway and promoted the progress of neuroblastoma.