The Study Of The Biological Behavior By MiR-155-5p And Target Gene IGF2 In Wilms Tumor

Objective: To investigate the expression of miR-155-5p in WT and the effect of its targeted regulation of IGF2 and mediated PI3 K signaling pathway on the biological behavior of WT cells.Methods: 1.Clinical samples analyses:(1)Differentially expressed miRNAs(DE-miRNAs)and mRNAs(DEGs)in WT blood and tissues were identified by using miRNA microarray and RNA-sequencing respectively;(2)RT-qPCR was used to explore miR-155-5p and IGF2 expression and their clinical significance in WT specimens;(3)Immunohistochemical staining,western blotting were performed to detect the expression of IGF2,PI3 K,AKT and mTOR.2.Bioinformatic analyses:(1)DE-miRNAs and DEGs in WT obtained from Gene Expression Omnibus(GEO)and Therapeutically Applicable Research to Generate Effective Treatments(TARGET)were identified by using the “edgeR” package;(2)Prediction and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analyses were used to predict thepotential functions of DE-miRNAs;(3)Gene Ontology(GO)and KEGG enrichment analyses were used to predict the potential functions and signal pathways with DE-mRNAs.3.Cytological experiments:(1)WT cell lines G401,SK-NEP-1 and normal renal tubular epithelial cell line HK2 were used to examine the expression of miR-155-5p;(2)Immunohistochemical staining,western blotting and dual-luciferase reporter assays were performed to study the mechanisms involved;(3)The CCK-8,flow cytometry,wound healing and transwell assays were performed to identify the effects of miR-155-5p and IGF2 knockdown on cell proliferation,apoptosis,migration and invasion,respectively.Results: 1.Compared with the normal control group,miR-155-5p was downregulated in cell lines,blood and tissues from WT patients who did not receive chemotherapy before surgery(P < 0.001)but was upregulated in tissues from WT patients who had received chemotherapy before surgery(P < 0.001).IGF2,PI3 K,AKT and mTOR were found to be upregulated in WT tissues(P < 0.001).Additionally,miR-155-5p and IGF2 were significantly correlated with TNM stage and lymphatic metastasis in WT patients(P < 0.05).2.Bioinformatics analysis showed that the expression of miR-155-5p and IGF2 in WT was consistent with the tissue experiment(P < 0.05).And IGF2 was predicted as a target gene for miR-155-5p.The KEGG pathwayenrichment analysis showed that these target genes are involved in some signaling pathways that are significantly correlated with tumor progression in WT,such as the PI3K-Akt signaling pathway,the Hippo signaling pathway,the mTOR signaling pathway,and the insulin signaling pathway.The KEGG disease enrichment analysis demonstrated that targets were associated with the cancers,congenital disorders and nervous system.Additionally,GO annotation of DE-mRNAs are focused on cellular component organization or biogenesis and process,binding,catalytic activity,tRNA transcription from RNA polymerase III promoter,DNA replication factor C complex,DNA endoreduplication and positive regulation of telomerase RNA localization to Cajal etc.The KEGG enrichment of DE-mRNAs mainly included cancers,RNA degradation,ribosome,pyrimidine metabolism,p53 signaling pathway,mRNA surveillance pathway,Hedgehog signaling pathway and cell cycle.3.Molecular mechanism exploration indicated that IGF2 was downregulated by miR-155-5p via direct binding to its 3'UTRs in WT cell lines.Furthermore,IGF2,PI3 K,AKT and mTOR expression was inversely correlated with miR-155-5p expression,and PI3 K,AKT and mTOR expression was positively correlated with IGF2 expression in cell culture.Functional studies demonstrated that miR-155-5p upregulation and IGF2 knockdown suppressed cell proliferation,migration and invasion and induced cell apoptosis.Moreover,the tumor-suppressing effects ofmiR-155-5p in cells were abrogated by miR-155-5p inhibitor treatment.Conclusion: 1.miR-155-5p expression is significantly reduced in WT blood samples,tissues and cells,but it is increased in post-chemotherapy WT tissues,and IGF2,PI3 K,AKT,mTOR are significantly increased in WT.The expression of miR-155-5p and IGF2 are significantly associated with TNM stage and lymphatic metastasis in WT patients.2.The KEGG pathway enrichment of DE-miRNAs target genes are PI3K-Akt signaling pathway and the mTOR signaling pathway.DE-mRNAs enrich signaling pathways on RNA degradation,p53 signaling pathway and cell cycle.3.miR-155-5p inhibits WT cell proliferation,migration,and invasion and promoted apoptosis through inactivating the PI3K/AKT/mTOR signaling pathway by binding and inhibiting IGF2.4.IGF2 functions as a tumor facilitator by accelerating cell proliferation,migration,and invasion and suppressing apoptosis in WT.5.The inhibitory role of miR-155-5p in WT progression and could facilitate the development of miRNA directed prognosis and therapeutics for WT patients.