MiR-146b-5p Inhibits T Cell Acute Lymphoblastic Leukemia Invasion Via Targeting IL-17A

Background and objective:T cell acute lymphoblastic leukemia(T-ALL)is a hematological neoplasm occurring in 10-15%of pediatric and 20-25%of adult cases of acute lymphoblastic leukemia.The pathogenesis of T-ALL is a multiple step oncogenic process associated with cancer cell proliferation,survival,invasion and infiltration.Increasing evidence suggests that miRNA dysfunction is involved in T-ALL tumorigenesis.This study aims to screen differentially expressed miRNA between T-ALL and control samples,and to study the biological functions and mechanisms of the differentially expressed miRNA and its target gene in T-ALL.Research contents and methods:Screening was performed to identify miRNAs that were differentially expressed between T-ALL and control samples in GEO data sets.Expression of differentially expressed miRNAs(miR-146b-5p)in T-ALL clinical samples and cell lines was verified by qPCR.MiRNA target genes were predicted by three online gene targeting tools(miRanda,miRWalk and Targetscan),and GeneAnalytics was used to analyze the role of miRNAs in tumorigenesis.Biological assays examining cell proliferation,apoptosis,cell cycle,cell migration and invasion were used to confirm the importance of dysregulated miRNAs in T-ALL.One of the potential target genes of miR-146b-5p(IL-17A)was validated by qPCR,Western Blot and dual luciferase reporter gene system.The role of IL-17A in T-ALL progression was detected by cell proliferation and transwell assay.IL-17A expression in T-ALL cell lines and clinical samples was detected by suspension array and qPCR/ELISA respectively.RT-PCR and flow cytometry were used to detect the expression of IL-17RA in T-ALL cell lines and clinical samples.The biological functions of IL-17A in T-ALL were analyzed by GSEA.Furthermore,T-ALL metastasis-related molecules mediated by IL-17A were confirmed by qPCR and Western Blot.IL-17A related signaling pathways in T-ALL were predicted by STRING analysis,and the effect of NF-κB signaling pathway related molecules was verified by Western Blot.The effects of IL-17A on the invasion and infiltration of T-ALL cells(MOLT-4)were studied in mouse model.The expression of CD7 in peripheral blood,liver and spleen cells of mice were detected by flow cytometry.The count of MDSC cells in liver and spleen tissue cells were analyzed by flow cytometry.Results:Bioinformatics analysis result showed that miR-146b-5p is a differential expressed gene between T-ALL cells(patients and cell lines)and normal T cells.GeneAnalytics results showed miR-146b-5p’s potential target genes correlated with tumorigenesis.miR-146b-5p expression was decreased in T-ALL patient samples and cell lines.Further examination demonstrated that over-expression of miR-146b-5p inhibited T-ALL cell migration and invasion.Subsequent target identification tools found a total of 145 potential target genes for miR-146b-5p,IL-17A was verified to be the key target gene of miR-146b-5p regulation.IL-17A was secreted and its receptor IL-17RA was expressed in T-ALL patients and cell lines.Furthermore,exogenous IL-17A significantly promoted T-ALL cell invasion after inhibition of endogenous IL-17A.GSEA analysis found that IL-17A played an important role in promoting invasion mainly through matrix metallopeptidases,and STRING analysis found that IL-17A and NF-κB1 have a very close relationship.Exogenous EL-17A could activate NF-κB-related signaling pathway,including the upregulation of MMP9.Expression of MMP9 was abolished after treatment with the NF-κB inhibitor PDTC.In the mouse model,the expression level of CD7 in peripheral blood and liver tissue of IL-17A-/-mice was decreased,while its expression in IL-17A-/-rescue cells increased.The expression of CD7 in mice spleen did not show any significant change.The results showed that IL-17A promoted MOLT-4 cells infiltration into mice liver.The count of MDSCs in spleen tissue of IL-17A-/-mice was the lowest,which suggests that IL-17A may promote MDSC cells infiltrating into mice spleen.Conclusions:1.miR-146b-5p is rarely expressed in T-ALL clinical samples and cell lines.Over expression of miR-146b-5p can inhibit invasion of T-ALL cells.2.IL-17A is the target gene of miR-146b-5p.3.IL-17A regulates T-ALL cells in an autocrine fashion.These cells highly express IL-17RA.IL-17A can promotes T-ALL cells invasion.4.IL-17A up-regulates MMP9 through NF-κB signaling pathway,which promotes T-ALL cells invasion.5.IL-17A promotes MOLT-4 cells infiltration into mouse liver in vivo.Moreover,IL-17A also promotes MDSC cells infiltration into the mouse spleen in vivo.