ABSTRACT
AIMS: Aberrant expression of microRNAs (miRNAs) is frequent in various cancers including gliomas. We aimed to characterize the role of miR-16-5p as a candidate tumour suppressor miRNA in gliomas. METHODS: Real-time PCR-based approaches were used for miRNA and mRNA expression profiling of glioma and non-neoplastic brain tissues as well as glioma cell lines. Protein levels were determined by Western blotting. In vitro analyses were performed following overexpression of miR-16-5p, trichostatin A (TSA) treatment, and siRNA-mediated knock-down of HDAC3 in glioma cells. Effects of miR-16-5p on glioma cell viability, apoptosis and response to irradiation and temozolomide (TMZ) were assessed. RESULTS: Expression of miR-16-5p was reduced relative to control brain tissue in isocitrate dehydrogenase (IDH)-mutant astrocytomas of World Health Organization (WHO) grades II, III and IV, and a subset of IDH-wildtype glioblastomas WHO grade IV. MiR-16-5p expression was lower in IDH-mutant than in IDH-wildtype gliomas, and down-regulated in IDH-wildtype glioma lines. MiR-16-5p overexpression reduced expression of important cell cycle and apoptosis regulators in glioma cells, including CDK6, CDC25A, CCND3, CCNE1, WEE1, CHEK1, BCL2 and MCL1. In line, CDK6, WEE1, CHEK1, BCL2 and MCL1 transcript levels were increased in WHO grade III or IV gliomas. TSA treatment and HDAC3 knockdown in glioma cells induced miR-16-5p up-regulation and reduced expression of its targets. Moreover, miR-16-5p overexpression inhibited proliferation and induced apoptosis in various glioma cell lines and increased sensitivity of A172 glioma cells to irradiation and TMZ. CONCLUSION: Reduced expression of miR-16-5p contributes to glioma cell proliferation, survival and resistance to cytotoxic therapy.
Subject(s)
Brain Neoplasms/genetics , Drug Resistance, Neoplasm/genetics , Gene Expression Regulation, Neoplastic/genetics , Glioma/genetics , MicroRNAs/genetics , Apoptosis/genetics , Brain Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/genetics , Down-Regulation , Glioma/pathology , HumansABSTRACT
All-trans retinoic acid is a potent promoter of cellular differentiation processes, which is used in cancer therapy. Glioblastoma spheroid cultures are enriched in tumor-initiating cells, and provide a model to test new treatment options in vitro. We investigated the molecular mechanisms of response to exposure to differentiation-promoting conditions in such cultures. Microarray analyses of five independent cultures showed that after induction of differentiation, inhibitors of transforming growth factor-beta/bone morphogenetic protein, Wnt/beta-catenin and IGF signaling were upregulated, whereas expression of several microRNAs decreased, particularly that of the miR-17-92 cluster. In primary astrocytic gliomas (n=82), expression of several members of miR-17-92 was significantly higher relative to those of normal brain (n=8) and significantly increased with tumor grade progression (P<0.05). A high-level amplification of the miR-17-92 locus was detected in one glioblastoma specimen. Transfection of inhibitors of miR-17-92 induced increased apoptosis and decreased cell proliferation in glioblastoma spheroids. Mir-17-92 inhibition was also associated with increased messenger RNA (mRNA) and/or protein expression of CDKN1A, E2F1, PTEN and CTGF. The CTGF gene was shown to be a target of miR-17-92 in glioblastoma spheroids by luciferase reporter assays. Our results suggest that miR-17-92 and its target CTGF mediate effects of differentiation-promoting treatment on glioblastoma cells through multiple regulatory pathways.