The Role And Molecular Mechanism Of MiR-92a In Glioma Cells And Glioma Stem Cells

Glioblastonma (GBM) is one of the most common primary brain tumor with high mortality and morbidity. Despite major advancements have been made in the surgery, postoperative chemotherapy and radiation therapy, the prognosis of patients suffering from malignant glioma remains depressed with an average survival time of about 15 month after diagnosis.Furthermore, a small subpopulation of cancer cells has been proved to be self-renewal and rulti-potential differential, namely glioma stem cells (GSCs). These cells are regarded as the main causes for tumor infiltration, migration, invasion, recurrence and resistance to radiotherapy and chemotherapy.MicroRNAs (miRNAs) are a class of highly conserved, small endogenous non-coding RNA molecules (?22 nucleotides), which regulate gene expression at the posttranscriptional level They suppress translation of protein and/or promote degradation of mRNA by base-pairing with complementary sequences in the 3' untranslated region (UTR) of downstream mRNAs. MicroRNAs has been widely reported to particpate in the tumor carcinogenesis as suppressors or promoters. Moreover,increasing evidences have proved that miRNA played a vital role in regulating the differentiation and self-renewal of cancer stem cells. Therefore,modulating miRNA expressions might be a promising therapeutic approach for malignant tumors.In this study, we found that miR-92a was significantly down-regulated in GSCs when compared to glioma cells. miR-92a was up-regulated in glioma cell lines and human glioma samples against the control group. Overexpression of miR-92a inhibited the proliferation but promoted the apoptosis of glioma cells. However,the role of miR-92a in the migration and invasion of glioma cells and in the survival, metastasis and self-renewal of GSCs still remained unclear. Therefore, fully understand the function and molecular mechanism of miR-92a in glioma cells and GSCs may provide a novel therapeutic strategy for glioma targeted therapy.Research purposesExplore molecular mechanism of miR-92a in glioma cells and GSCs and find clues for novel therapeutic strategies against glioma.Methods1. Isolate GSCs from glioma cells by serum-free culture and identify target cells by immuno fluorescence staining.2. Analyze the miRNA expression profiles of glioma cell line (U87) and glioma stem cells(U87s).3. Detect the role of miR-92a in glioma's proliferation, metastasis and apoptosis by MTT assay,wound healing assay, Transwell fliers and Annexin V-FITC/PI staining.4. Assess the effect of miR-92a on tumorgenesis in vivo.5. Evaluate the function of miR-92a in the survival, migration and self-renewal of GSCs by tumor sphere formation assay, migration assay and Western blot.6. Predict the potential targets of miR-92a by bioinformatics' method and confirm with dual luciferase reporter system7. Investigate the molecular mechanism of miR-92a in glioma cells and GSCs by Western blot and immunofluorescence staining.Results1. GSCs were enriched from U87 and U251 cell lines by serum-free culturing. The immunofluorescence staining indicated that CD133 (cancer stem cells marker) and nestin(neural stem cells marker) were co-expressed on the cell surface of GSCs. When cultured in the medium supplement with fetal bovine serum, the tumor spheres would differentiate and express glial fibrillary acidic protein (GFAP, astrocyte's maker) and ?-tubulin-? (neuron marker).2. miRNAs expression profile indicated that the expression of 94 miRNAs differed between U87 and U87s. qPCR analysis revealed that miR-92a was down-regulated in U87s and U251s cells as compared to U87 and U251 cells.3. Depleting endogenous miR-92a inhibited the proliferation, migration and invasion, and promoted the apoptosis of U87 and U251 cells in vitro.4. Suppression of miR-92a prevented the xenograft growth of glioma in vivo.5. Overexpression of miR-92a impaired the survival, metastasis and tumor spheres formation of GSCs. In addition, up-regulation of miR-92a led to a significant decrease in CD133 and Nestin. When compared with the negative control group, the diameter of tumor spheres decreased slightly after transfected with miR-92a mimics.6. The results from bioinformatics and dual Luciferase reporter system revealed that miR-92a targeted CDH1 in glioma cells and binded to the 3'UTR of Notch-1 in GSCs.7. Western blot and immunofluorescence staining proved that down-regulation of miR-92a led to a robust increase in CDH1 transcriptionally and translationally, and subsequently suppressed the expression of nuclear ?-catenin. In contrast, total P-catenin expression remained unchanged in U87 and U251 cells.8. Increasing miR-92a expression induced a considerable decrease in transcriptional and translational levels of Notch-1 and reduced the phosphorylation level of Akt in GSCs. Total Akt were barely affected.9. Up-regulation of CDH1 dramatically inhibited the metastasis ability of glioma cells while cell proliferation and apoptosis remained the same. Moreover, knockdown of Notch-1 repressed the survival, migration and self-renewal of GSCs.ConclusionsIn the present study, the high-throughput analysis of miRNAs expression indicated that miR-92a expression differed between glioma cells and GSCs from glioma cells. Inhibition of miR-92a markedly suppressed glionma cells nalignance in vitro and decreased the grtowth of xenograft tumors in vivo. Overexpression of miR-92a repressed the growth, miration and sternness of GSCs. In terms of mechanism, miR-92a targeted CDH1 and Notch-1, and subsequently affected the ?-catenin and Akt signaling pathways in a context-dependent manner.This study revealed that miR-92a might play a critical role in the development of glioma.