Tumor suppressive miRNA-34a suppresses cell proliferation and tumor growth of glioma stem cells by targeting Akt and Wnt signaling pathways.

MiRNA-34a is considered as a potential prognostic marker for glioma, as studies suggest that its expression negatively correlates with patient survival in grade III and IV glial tumors. Here, we show that expression of miR-34a was decreased in a graded manner in glioma and glioma stem cell-lines as compared to normal brain tissues. Ectopic expression of miR-34a in glioma stem cell-lines HNGC-2 and NSG-K16 decreased the proliferative and migratory potential of these cells, induced cell cycle arrest and caused apoptosis. Notably, the miR-34a glioma cells formed significantly smaller xenografts in immuno-deficient mice as compared with control glioma stem cell-lines. Here, using a bioinformatics approach and various biological assays, we identify Rictor, as a novel target for miR-34a in glioma stem cells. Rictor, a defining component of mTORC2 complex, is involved in cell survival signaling. mTORC2 lays downstream of Akt, and thus is a direct activator of Akt. Our earlier studies have elaborated on role of Rictor in glioma invasion (Das et al., 2011). Here, we demonstrate that miR34a over-expression in glioma stem cells profoundly decreased levels of p-AKT (Ser473), increased GSK-3ß levels and targeted for degradation ß-catenin, an important mediator of Wnt signaling pathway. This led to diminished levels of the Wnt effectors cyclin D1 and c-myc. Collectively, we show that the tumor suppressive function of miR-34a in glioblastoma is mediated via Rictor, which through its effects on AKT/mTOR pathway and Wnt signaling causes pronounced effects on glioma malignancy.

MiR-145 functions as a tumor-suppressive RNA by targeting Sox9 and adducin 3 in human glioma cells.

BACKGROUND: MicroRNAs (miRNAs) are increasingly being recognized as being involved in cancer development and progression in gliomas. METHODS: Using a model cell system developed in our lab to study glioma progression comprising human neuroglial culture (HNGC)-1 and HNGC-2 cells, we report here that miR-145 is one of the miRNAs significantly downregulated during malignant transformation in glioblastoma multiforme (GBM). In a study using tumor samples derived from various glioma grades, we show that expression of miR-145 is decreased in a graded manner, with GBM patients showing lowest expression relative to lower-grade gliomas (P < .05) and normal brain tissues (P < .0001). Functional studies involving ectopic expression of miR-145 in glioma cells had a negative impact on cell proliferation and tumor development, as well as invasion and induced apoptosis, providing further support to the concept that inactivation of miR-145 is important for glioma disease pathogenesis. More notably, these growth-suppressive effects of miR-145 are mediated through its target proteins Sox9 and the cell adhesion-associated molecule adducin 3 (ADD3). RESULTS: Inhibiting Sox9 and ADD3 rescued effects of miR-145 loss. Interestingly, miR-145 loss in glioma cells led to overexpression of molecules involved in cell proliferation, like cyclin D1, c-myc, and N-myc, as well as enhanced expression of cell adhesion- and invasion-related molecules N-cadherin and E-cadherin, an effect which was again restored upon miR-145 overexpression in glioma cells. The miR-145 promoter was methylated at its cytosine-phosphate-guanine (CpG) islands in the glioma cell lines studied. CONCLUSION: Our study demonstrates that miR-145 has a tumor-suppressive function in glioblastoma in that it reduces proliferation, adhesion, and invasion of glioblastoma cells, apparently by suppressing the activity of oncogenic proteins Sox9 and ADD3. Reduced levels of miR-145 may lead to neoplastic transformation and malignant progression in glioma due to unregulated activity of these proteins.

Expression of nestin--a stem cell associated intermediate filament in human CNS tumours.

BACKGROUND & OBJECTIVES: Nestin is an intermediate filament protein expressed in undifferentiated cells during the development of brain and is considered as a marker for neuroepithelial stem cells. Expression of this protein in various CNS tumour cells suggests the possibility of existence of tumour stem cell modulating the evolution. We carried out an immunohistochemical study to demonstrate the expression of nestin and its co-expression with neuronal and glial intermediate filament and correlate with the grade of malignancy. METHODS: Formalin fixed, paraffin processed sections from two human foetuses, 16 brain tumours of both neuronal and glial lineage and two metastatic tumours were immunostained with polyclonal antibody to nestin. Serial sections from primary brain tumours were also stained with monoclonal antibody to neurofilament (NF) and glial fibrillary acidic protein (GFAP). Fluorescent double labeling was carried out on four cases using laser confocal microscopy, to document co-localization of nestin with other intermediate filaments in the tumour cells. RESULTS: Nestin expression was observed along the paraventricular zone of human foetuses and in brain tumours of both glial and neuronal lineage, of both high and low grades of malignancy. In addition, mature dysplastic spinal motor neurons adjacent to tumour and cerebellar Purkinje cells also expressed nestin along with neurofilament. INTERPRETATION & CONCLUSION: Nestin expression was noted in both low and high grade brain tumours and dysplastic neurons and did not parallel the malignant grade of the tumour. The expression of nestin in tumour cells and dysplastic neurons suggests aberrant expression of antigenically primitive proteins in cells to facilitate remodelling of the cell and migration. More studies are needed to elucidate the concept.