MicroRNA 335 Is Required for Differentiation of Malignant Glioma Cells Induced by Activation of cAMP/Protein Kinase A Pathway

Glioma is the most common malignant cancer affecting the central nerve system, with dismal prognosis. Differentiation inducing therapy is a novel strategy that has been preliminarily proved effective against malignant glioma. We have reported previously that activation of cAMP/ protein kinase A (PKA) pathway is capable of inducing glioma cell differentiation, characterized by astrocyte- like shape and dramatic induction of astrocyte biomarker glial fibrillary acidic protein (GFAP). However, little progress has been made on molecular mechanisms related. Here we demonstrate that microRNA 335 (miR-335) is responsible for the glioma cell differentiation stimulated by activation of cAMP/ PKA pathway. In the cAMP elevator cholera toxin-induced differentiation model of rat C6 glioma cells, miR335 was significantly up-regulated, which was mimicked by other typical cAMP/ PKA pathway activators (e. g., forskolin, dibutyryl-cAMP) and abolished by PKA-specific inhibitor (9R, 10S, 12S)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl1- oxo-9,12-epoxy-1H-diindolo[1,2,3-fg: 3',2',1'-kl] pyrrolo[ 3,4-i] [ 1,6] benzodiazocine-10-carboxylic acid, hexyl ester (KT5720). In an assay measuring gain and loss of miR-335 function, exogenetic miR-335 resulted in induction of GFAP, whereas miR-335 specific inhibitor antagomir-335 violently blocked cholera toxin-induced GFAP up-regulation. It is noteworthy that in human U87-MG glioma cells and human primary culture glioma cells, miR-335 also mediated cholera toxin-induced differentiation. Taken together, our findings suggest that miR-335 is potently required for differentiation of malignant glioma cells induced by cAMP/ PKA pathway activation, and a single microRNA may act as an important fate determinant to control the differentiation status of malignant gliomas, which has provided a new insight into differentiation-inducing therapy against malignant gliomas.