| Glioma represents the most common malignant tumor type in the centralnervous system, which accounts for41-44.6percent of intracranial tumors. Thistype of tumor is characteristic of high mortality and morbidity and highrecurrence rate. Patients diagnosed with the most malignant subtype,glioblastoma multiforme, only have a median survival time of no more than14months. Although remarkable progresses have been made on the availabletreatments for gliomas including surgery, chemotherapy and radiotherapy, theprognosis of patients is still dismal, which is mainly attributed to its inherentlyprogressive overgrowth, and expansively diffuse and invasion of glial tissues.Therefore, there is an urgent need for identification of molecular targets to curethis disease. MicroRNAs (miRNAs) are a class of endogenous and smallnon-coding RNAs, which are22nucleotides in length and mainly regulate theexpression of target genes at the post-transcriptional level. The role of miRNAsin the tumorigenesis has been extensively studied and it sheds light on thepotential application as an important adjuvant treatment. However, relatively less miRNAs have been known in gliomas up to now. In this study, we identifiedmiR-483-5p as a novel down-regulated miRNA in human glioma andinvestigated its role and mechanism in the cellular proliferation of gliomas asfollows.Firstly, through analyzing the miRNA-array profiles of three pair specimens ofhuman glioblastoma and adjacent normal brain, we found miR-483-5p wassignificantly down-regulated by100fold among91deregulated miRNAs ingliomas, which was subsequently confirmed in nine human glioma samples withdifferent grades and three human glioma cell lines. The role of miR-483-5p in thetumorigenesis is unclear. Moreover, our results imply potential involvement ofmiR-483-5p in the gliomagenesis. Thus, we further explored the function ofmiR-483-5p in gliomas.We chose U251and U87cells for in vitro study based on their relative highand low expression levels of miR-483-5p. For overexpression of miR-483-5p inglioma cells, we firstly constructed lentiviral expression plasmids carryingpre-miR-483-5p and its flanking sequences. Then miR-483-5p-expressing andcontrol lentiviruses were respectively packaged in HEK293T cells. U251andU87cells were infected with above lentiviruses and selected with blasticidin.Finally, after confirming the efficiency of the stable transfection, we investigatedthe influence of miR-483-5p-overexpression on the cellular growth and cell cycledistribution by the method of MTT and flow cytometric analysis. Our resultsshowed a decreased growth of cells overexpressing miR-483-5p compared withthe control cells (P<0.01), which was also companied with an increase of cellproportion in the G0/G1phase (P<0.01) and a corresponding decrease in the Sand G2/M phase. To inhibit the expression of miR-483-5p, U251cells weretransfected with miR-483-5p inhibitors (anti-miR-483-5p). The increased cell number induced by anti-miR-483-5p was observed at48h post-transfection(P<0.01). Taken together, these results indicate that miR-483-5p inhibited cellularproliferation of gliomas at least through arresting cells in the G0/G1phase.In order to investigate the molecular mechanism underlying the growthinhibition by miR-483-5p, we further identified its target genes involved intumorigenesis. Through analyzing predicted targets databases calculated by thealgorithm of TargetScan and miRanda, we got six potential target genesassociated with the cellular proliferation and migration of tumors, among whichthe relative luciferase activities of3’-UTRs and mRNA expression of ERK1(MAPK3), ALCAM and PLA2G5were significantly suppressed by miR-483-5p(P<0.05). ERK1is a key member of the MAPKs (mitogen-activated proteinkinase) family, which regulates cellular proliferation in tumors. As the mostinterested target gene, the reporter plasmid of mutated ERK13’-UTR wascontructed and the endogenous ERK1expression was examined in the cellstransfected with ERK1-expressing plasmid or anti-miR-483-5p. We found thatthe decreased luciferase activity induced by miR-483-5p was restore when theconstruct of wild-type ERK13’-UTR was replaced by the mutated one.Additionally, compared with control, the mRNA and protein expression of ERK1was reduced in U87cells overexpressing miR-483-5p (P<0.05), while increasedin U251cells transfected with anti-miR-483-5p (P<0.01). These data demonstratethat ERK1is directly regulated by miR-483-5p at the post-transcriptional level.To investigate whether the growth suppression induced by miR-483-5poverexpression in glioma cells was due to inhibited ERK1expression, U251cellswere co-transfected with ERK1siRNA and anti-miR-483-5p. Our results showedthat transfection of anti-miR-483-5p promoted glioma cell proliferation andupregulation of ERK1expression, whereas transfection of ERK1siRNA decreased the number of glioma cells and the expression level of ERK1. However,after co-transfection, the cell number and ERK1level induced byanti-miR-483-5p were reversed. These results suggest that miR-483-5p inhibitsglioma cell proliferation at least partly through regulation of ERK1.1.5×10~6U87-control and U87-miR-483-5p cells were respectively andsubcutaneously inoculated in both sides of flanks of nude mice to establishxenograft models of human glioma. The tumor growth was observed andexamined according to the tumor volume measured termly. The animals weresacrificed and the tumor was weighted at the24th day after inoculation. Theresults showed that tumor grew slowly, both tumor volume and tumor weightsignificantly decreased in U87-miR-483-5p group as compared with those inU87-control group (P<0.05). This part of work suggests that in vivo tumorformation was inhibited by overexpression of miR-483-5p.In summary, we identified a novel down-regulated miRNA miR-483-5p inhuman gliomas and found its role in the inhibition of cellular proliferation andilluminated its underlying molecular mechanism. miR-483-5p may serve as apotential target for diagnosis and treatment of gliomas. |
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