The Mechanism Of Wilms' Tumor Proliferation And Invasion Via TGF-?/Smad Signaling Pathway

Objective: Wilms Tumor(WT)is a common malignant embryonal tumor in children.The pathogenesis is still unclear.The causes of WT may involve genetic changes,chromosomal abnormalities,and genetic factors.In recent years,studies have shown that the abnormal expression of TGF-?1 in WT is related to tumor metastasis,recurrence and cell invasion ability.However,the exact mechanism remains unclear.This study intends to explore the TGF-?/Smad expressed in WT and explore the differences expression genes via TCGA and GEO database and find TGF-?1mediated downstream gene expression.Meanwhile,in order to clarify the carcinogenic and provide a new treatment strategy of WT,the intervention of TGF-?1 receptor and downstream genes used WT cell and animal models.Method: Part I: TGF?/Smad protein expression in WT was detected and its correlation with clinical data was discussed.The effect of TGF?/Smad signal inhibition on WT cells and animal models was detected:(1)TGF?1 and p-Smad2/3 expression levels were detected by immunohistochemical staining in 60 WT and adjacent control tissue samples;(2)CCK-8,wound healing assay,invasion assay and flow cell apoptosis assay were used to detect the effects of T?RI inhibitor and TGF? agonist on the proliferation,migration,invasion and apoptosis of G401 cells.(3)WT animal model was established to detect the effect of T?RI inhibitor on tumor cell proliferation and the effect of the inhibitor on liver and kidney function metabolism.Part II: Screening of core genes in the GEO and TCGA databases:(1)R Studio software was used to find the differentially expressed genes in the GEO dataset(GSE66405,GSE73209)and TCGA-target-to-WT transcriptome sequencing results,and the up-regulated genes in the three datasets were found.(2)David was used to analyze tumor signaling pathways enriched in up-regulated and down-regulated genes.(3)Cytoscape software and String were used for protein interaction network analysis to find the core genes,and GSEA was used to analyze the upstream genes regulated by the core genes.(4)The expression of the core genes in nephroblastoma was verified by immunohistochemical staining;(5)Designed the si-RNA model,CCCK8,wound healing and cycle assay were constructed to detect its effects on proliferation,migration and cell cycle regulation of WT cells.Part III: To detect the correlation of TGF?1,c-Myc and CDC20 expression in WT,to inhibit the TGF? signaling pathway in WT,and to detect the effect of TGF?1,c-Myc and CDC20 expression in the downstream MYC/CDC20 mediated cell cycle and EMT:(1)WB was used to detect the expression of TGF?1,c-Myc and CDC20 in the tumor tissues of nephroblastoma,and Spearman was used to analyze the correlation of protein expression.(2)T?RI inhibitor was used to intervene WT cell line and tumor-bearing mouse model,and the expression of downstream EMT-related proteins(E-CAD,N-CAD,?-CAT,CK)was detected by WB test and IP test.(3)Ras protein inhibitors were used to intervene WT cell lines to detect the expression of cell cycle-related proteins;(4)After the CDC20 gene in G401 and SK-NEP-1 was silenced,the cell cycle-related protein expression was detected.Results: Part I:(1)TGF-?1,p-Smad2/3 protein expression was significantly increased in WT,and was significantly correlated with clinical stage and tumor invasion and metastasis.(2)Inhibition of TGF-?1 receptor I can significantly reduce proliferation,migration and invasion of WT cells,and increase the ability of tumor apoptosis;(3)In vivo,T?RI inhibitor can significantly reduce the proliferation of tumor tissue,and the inhibitor has no liver and kidney damage.Part II :(1)The study in this part found that GSE66405,GSE73029,TCGA-target-WT together had 44 up-regulated genes and 272up-regulated genes,and the up-regulated genes were mainly enriched in the Cell cycle and Cell division cycle.(2)Protein interaction network analysis of Top linked genes revealed CDC20 core with more associations,which was mainly regulated by upstream genes such as MYC.(3)Transfection of si-CDC20 into WT cell lines G401 and SK-NEP-1 could significantly inhibit the proliferation and migration of tumor cells.Meanwhile,the cells were stagnated in the G2/M phase.Part III :(1)TGF?1,c-myc and Cdc20 expression were increased in WT,and the three were positively correlated;(2)Inhibition of Ras could significantly reduce the expression of c-myc,CDC20,and increase the expression levels of G2/M phase proteins(Securin and Cyclin B1 and Cyclin A).(3)Inhibition of T?RI decreased the expression levels of N-CAD,SMA,MMP7,?-CAT,and increased the expression levels of E-CAD and CK.(4)Si-Cd C20 significantly increased the expression levels of G2/M phase proteins(Securin and Cyclin B1 and Cyclin A)in WT cells.Conclusion: Activation of TGF-?/Smad signaling pathway in WT may be the cause of proliferation,metastasis and invasion.Inhibition of TGF-?/Smad signaling pathway can significantly reduce the proliferation,invasion and migration of WT.The mechanism may be related to the regulation of TGF-?/Smad signaling pathway in Myc/ Cdc20,resulting in cell cycle disturbance and TGF?-mediated EMT.