MicroRNA-21 Contributes To VM-26 Resistance In Glioblastoma By Targeting LRRFIP1

MicroRNA are a class of 18-26nt noncoding RNAs that negatively regulate the expression of target genes by interacting with complementary sites in the 3' untranslated region(3'UTR).This relatively small number of regulatory RNA molecules is predicted to regulate the expression of at least 30%of human transcripts,including those involved in cancer.Several research groups have provided evidences that miRNAs control a wide array of biological processes including early development,cell proliferation and cell death,stress reaction,apoptosis and fat metabolis,cell differentiation,and brain development.The close relationship between miRNAs and cancer was also been demonstrated.Many miRNAs have been classified to oncomiRs and suppressor genes in specific cancers.Mounting evidence indicates that MicroRNA-21(miR-21) is overexpressed in many cancers including glioblastoma and might play a fundamental role as oncogene in tumorigenesis.As a members of the miR-17-92 cluster,miR-21 was shown to be associated with cell proliferation,apotosis,migration and drug resistant and contributes to tumor resistance to chemotherapy.Such as,The introduction of complementary oligonucleotides into glioma cells with high miR-21 levels leads to reduction of glioma cell viability associated with an increase in activity of caspases.The synergistic cytotoxicity of miR-21 knockdown and S-TRAIL in glioblastoma therapy has been found.These facts lead us to ask Whether miR-21 is also involved in the resistance to chemotherapeutic agents in treatment of glioblastoma.We investigated whether miR-21 mediated chemoresistance to the chemotherapeutic agent VM-26 in glioblastoma cells and sought to identify the candidate target genes for miR-21 by gene expression profiling.In order to examine the effect of miR-21 on glioblastoma cell proliferation.U373MG cells were transfected with miR-21ASO or control ASO and cell viability was detected by MTT assay.In U373MG cells,miR-21 ASO showed a significant antiproliferation effect compared with the control group,suggesting that miR-21 could accelerate glioblastoma cell proliferation.To explore the role of miR-21 in chemoresisitance of glioblastoma,U373MG cells were transfected with either miR-21 ASO or control ASO,and were subsequently incubated with different doses of VM-26.Then the cells were examined for viability levels by MTT assay.The result indicated that knockdown of miR-21 enhanced VM26-induced cell viability reduction in U373MG cells,and the sensitization effect of miR-21 inhibition showed a drug dose-dependent manner,suggesting that miR-21 could suppress chemo-sensitivity of glioblastoma。miR-21 usually affect cell phenotype through negatively regulating the expression of specific target genes.To explore the mechanism involving in miR-21-mediated drug resistance in glioblastoma,We selected combined strategies to identify new target gene which may participate in miR-21-mediated drug resistance.Frist step was searched candidate target genes of miR-21 through several bioinformatic database.Such as MIRANDA,TARGESCAN and PICTAR.Second,the 7267-gene microarray was applied to investigate the gene expression profile of miR-21 ASO treated U373MG cells comparing with control cells.Combining target prediction bioinformatics with expression profiling.LRRFIPl,which was up-regulated in miR-21 ASO treated cells,was predicted as a candidate target gene of miR-21.To determine the effect of miR-21 on LRRFIP1 mRNA and protein expression level, U373MG cells were transfected with miR-21 ASO or control ASO,and the mRNA level and protein level of LRRFIP1 was detected.The result indicated that suppression of miR-21 significantly increased expression of LRRFIP1 in both mRNA level and protein level.To confirm the specificity of this regulation,U373MG cells were transfected with EGFP report vector along with either miR-21 ASO or control ASO,the expression level of EGFP is obviously increased when endogenous miR-21 was blocked.However,when the reporter vector containing a mutational miR-21 binding site was used in the fluorescent reporter assay,the miR-21 ASO mediated fluorescent increase could no longer be detected. It is revealed that LRRFIP1 was directly regulated by miR-21.LRRFIP,also known as TRAF-interacting protein(TRIP),is a signaling component of the TNFR(tumor necrosis factor receptor) superfamily and contains a RING finger motif and an extended coiled-coil domain.TRIP interacts with the receptor/TRAF(TNF receptor-associated factor) signaling complex,and inhibits the TRAF2-mediated NF-κB activation.Activation of NF-κB is required for cell activation and also for protection against apoptosis and is a principal mechanism of tumor chemoresistance,which is primarily mediated by its antiapoptotic activity.Constitutive NF-κB activation has been described in various tumors,where it has been implicated in conferring chemoresistance. Additionally,inhibition of NF-κB signaling has been reported to sensitize tumor cells to apoptosis induced by TNF-αor anticancer agents.Here we find that suppression of miR-21 could up-regulate the expression of its target gene LRRFIP1,which inhibits NF-κB activation and leads to enhancement of VM26-mediated cell death in glioblastoma cells. This research is the first to show the chemosensitivity associated miR-21 in glioblastoma.MiR-21 contributes to VM-26 drug resistance through excessive depression of LRRFIP1 gene,leading to reduction of the cytotoxicity of chemotherapy drugs through activation of the NF-κB pathway.Our findings suggest that miR-21 represents a promising target for therapeutic manipulation to increase the efficacy of chemotherapeutic agents in treating glioblastoma.Since the partern of the interaction between cells and moleculars is complex and The express and fuction models of miRNA is diverse.more detail works are needed to evaluate the mechanisms miRNA-mediated drug resistance.