The Study On The Modulation Of GLDC And Malignant Phenotype In Gliomas By MicroRNA-874

[Background and Aims]MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression.MicroRNAs are involved in many physiological and pathological progress.Recent evidence indicates that special miRNAs may functions as one oncogenes or tumor suppressors and play a critical role in cancer initiation and progression by negatively regulating their target genes.Using RT-PCR,we found that hsa-miR-874 was significantly down-regulated in human glioma tissue samples.In this study,we focused on the effects of hsa-miR-874 on the phenotypes of glioma cells as well as the identification of the direct target genes of hsa-miR-874,in order to illuminate the molecular mechanisms of hsa-miR-874 in the initiation and progression of glioma.[Methods]We identified the results of previous glioma array by detecting and comparing hsa-miR-874 between in normal brains and gliomas with semi-quantitative RT-PCR.We detected hsa-miR-874 in glioma cell lines(LN229?U87?NB19?U251?LN308).Subsequently,We detected viability of cell by MTT assay and Colony-forming Assay,transfer ability by Transwell Assay,cell cycle changes by Flow Cytometer.TargetScan 5.1?STRING?protein interaction networks?KEGG grid?miRNA and RNAhybrid were used to pretect target gene of hsa-miR-874.Changes of hsa-miR-874 and its target gene were detected after transfecting hsa-miR-874 mimics in glioma cell lines(LN229?U87)by RT-PCR.The reliability of the direct target genes was confirrned by fluorescent reporter assay.Furthermore,the mRNA levels and protein levels of target genes in hsa-miR-874 inhibited glioma cells or tissues were detected with semi-quantitative RT-PCR and Western blot,in order to confirm the regulating role of hsa-miR-874 in target gene expression.[Results]Hsa-miR-874 expressed low in glioma cells than in normal brain tissues ma.LN229?U87 cell lines expressed hsa-miR-874 lower than other glioma cell lines.We report that after suppression of hsa-miR-874,the LN229?U87 cells proliferation activity and the colony formation activity were both inhibited.The cells after transfected presented G0/G1 cycle arrest and cells increase in S-phase fraction.Subsequently,we identified tumor suppressor GLDC as one of the candidate target genes for hsa-miR-874.The GLDC mRNA 3'untranslated region(3'UTR)contains the potential binding site of hsa-miR-874.The fluoresescent reporter assay also confirmed that hsa-miR-874 can directly bind to the specifie site of prohibitin mRNA 3'UTR and negatively regulate the gene expression.When hsa-miR-874 function was inhibited in glioma cells or tissues,mRNA level and protein level of GLDC were both reduced.[Conelusions]Hsa-miR-874 expressed low in glioma cell lines and glioma tissues.GLDC gene was identified to be target gene of hsa-miR-874 by fluoresescent reporter assay and Western blot test.Our results indicate that in glioma cells,miR-874 funetions as an tumor suppresser gene and inhibit cell proliferation.The over-expressed miR-874 in glioma cells can direetly down-regulate the expression level of oncogene prohibitin and result in the loss-of-function of prohibitin,leading to the activation of cell proliferation.The elucidation of common mechanisms of miR-874 in gliomas helps us to further understand caneer initiation and progression,and provides new clues to cancer diagnosis and therapy.