Hypoxia-inducible miR-196a modulates glioblastoma cell proliferation and migration through complex regulation of NRAS

Abstract

Background: Glioblastoma (GBM) is the most common and aggressive malignant brain tumor in humans. Hypoxia has been correlated with the aggressive form of glial tumors, poor prognosis, recurrence and resistance to various therapies. MicroRNAs (miRNAs) have emerged as critical mediators of hypoxic responses and have shown great potential for cancer diagnostics and therapeutics. Here, we focus on the regulatory and functional characterization of miR-196a, a hypoxia-inducible miRNA, in GBM.

Methods: Hypoxia/HIF regulation of miR-196a was assessed by RT-qPCR, promoter-luciferase and ChIP assays in GBM cell lines. miR-196a levels were analyzed in The Cancer Genome Atlas (TCGA)-GBM, Chinese Glioma Genome Atlas (CGGA) and Indian GBM patient cohorts. miR-target interactions were studied using RNA/protein quantification and 3'UTR luciferase assays. The effect of miR-196a overexpression/inhibition was assessed on cellular viability, migration and apoptosis under hypoxia and normoxia. Microarray-based gene expression profiling studies were performrd to study the effect of miR-196a on the GBM cellular transcriptome under hypoxia.

Results: We identified miR-196a as a hypoxia-inducible and hypoxia-inducible factor (HIF)-regulated miRNA that plays an oncogenic role in GBM. miR-196a was found to be significantly up-regulated in TCGA-GBM, CGGA glioma as well as Indian GBM patient cohorts. miR-196a overexpression was found to induce cellular proliferation, migration, spheroid formation and colony formation and to inhibit apoptosis, while miR-196a inhibition using anti-miR-196a yielded opposite results, suggesting an oncogenic role of miR-196a in GBM. We further unveiled NRAS, AJAP1, TAOK1 and COL24A1 as direct targets of miR-196a. We also report a complex competitive regulation of oncogenic NRAS by miR-196a, miR-146a and let-7 in GBM. Analysis of microarray-based gene expression data obtained by miR-196a inhibition under hypoxia revealed a role of miR-196a in HIF, calcium adhesion, Wnt and cell adhesion pathways. Interestingly, miR-196a was found to positively regulate the expression of various genes involved in the induction or stabilization of HIFs and in maintenance of hypoxic conditions, thereby suggesting the existence of an indirect miR-196a/HIF positive feedback loop under hypoxia.

Conclusions: Overall, our work identifies a novel association between hypoxia/HIF signalling and miR-196a in GBM and suggests its therapeutic significance.

Keywords: Glioblastoma; HIF-1; Hypoxia; NRAS; miR-196a.