The Role Of S100A8 And S100A9 In Proliferation And Migration Of Neuroblastoma SH-SY5Y Cells And Its Mechanism

Objective: Neuroblastoma(NB)is the second most common solid tumor with high malignancy in infants and children accounting for 6%~8% of pediatric malignancies.S100A8 and S100A9 are two important members of the S100 family of calcium-binding proteins.S100A8 and S100A9 are closely relatd to the occurrence and development of various tumors.However,their expression and mechanism of action in neuroblastoma have been rarely studied.This article explores the effects of S100A8 and S100A9 on proliferation and migration of neuroblastoma cells and their biological mechanisms.Methods: Bioinformatics analysis showed that S100A8 and S100A9 are two proteins closely related to the occurrence and development of neuroblastoma.The recombinant plasmid carrying the gene of interest and its vector plasmid were transfected into SH-SY-5Y cells of neuroblastoma by constructing a recombinant plasmid labeled with green fluorescent protein maker,respectively,to obtain stable cell lines with high expression of S100A8,S100A9 and low expression of S100A8 and S100A9.The recombinant plasmids included overexpression group: S100A8-SBI-piggbac,S100A9-SBI-piggbac and low-expression group: shRNA-S100A8(shS100A8 or shA8)and shRNA-S100A9(shS100A9 or shA9).The proliferation capacity of cells in the experimental group and the control group were detected by plate cloning experiment and MTT assay,and the cell viability was measured.Scratch test and Transwell were used to detect changes in cell migration ability.Sh-SY5 Y and its stable cell lines were treated with adenovirus-mediated RFP,GFP,Si-β-catenin and β-catenin,respectively,and the migration ability of cells was detected by scratch test.The plate cloning assay was used to detect the changes of cell viability and proliferation ability of the experimental group and the control group.Results:(1)Experiments by MTT assay and colony formation assay showed that in NB cell SH-SY5 Y,the proliferation of NB cells overexpressing S100A8(98±1.732)and S100A9(107±4.583)was significantly improved compared with the control group(69.333±6.11)(t=23.81,14.62,P<0.01).After S100A8(37.333 2.081)and S100A9(37 1.732)were knocked down in SH-SY5 Y cells,the proliferation activity of the significantly lower than that of the control group(t=19.35,24.14,P<0.01).(2)Scratch test and Transwell invasion and migration experiments showed that overexpression of S100A8(499.667±13.65)and S100A9(564.333±28.937)significantly increased cell migration and colony formation compared with the control group(360 ±20.075)(t=6.426、7.517,P<0.01).After knocking down S100A8(284±39.837)and S100A9(284±39.837),the migration rate of NB cells was significantly inhibited,and the formation of colonies was significantly reduced(t=5.364,5.848,P<0.01).(3)Experimental examination by scratch test,plate cloning and other experiments showed that the overexpression of β-catenin(91±2.828)significantly increased the cell migration rate and colony formation compared with the control group(Blank-Ad-GFP,75.5±3.536)(t=5.091,P<0.05).Compared with the control group(Blank-Ad-RFP,66.5±4.95),the knockout of β-catenin(57.5±3.536)significantly decreased the cell migration rate and colony formation(t=4.841,P<0.05).By comparing S100A8-Siβ-catenin(71±1.414)with S100A8-AdRFP(88±4.242),S100A9-Siβ-catenin(69±4.242)with S100A9-AdRFP(90±2.828),it was found that knockdown of β-catenin in NB cells reduced the migration rate and colony formation of SH-SY5 Y cells induced by S100A8 and S100A9(t=5.376、5.824,P<0.05).Conclusion:(1)Overexpression of S100A8 and S100A9 promoted the survival and migration of NB cells.(2)In NB cells,overexpression of S100A8 and S100A9 is consistent with the overexpression of β-catenin,which promotes the proliferation and migration of NB cells.(3)Knockdown of β-catenin can partially eliminate the ability of overexpression of S100A8 and S100A9 to induce proliferation and migration of NB cells,so S100A8 and S100A9 are closely related with β-catenin.Therefore,it is speculated that S100A8 and S100A9 may participate in the regulation of Wnt/β-catenin signaling pathway and promote the proliferation and migration of neuroblastoma by regulating the level of β-catenin in cells.