MicroRNA-149 is epigenetically silenced tumor-suppressive microRNA, involved in cell proliferation and downregulation of AKT1 and cyclin D1 in human glioblastoma multiforme

Abstract

Aberrant DNA methylation has been shown to inactivate tumor suppressor genes during carcinogenesis. MicroRNA-149 (miR-149) was recently demonstrated to function as a tumor suppressor gene in glioblastoma multiforme (GBM). However, the potential linkage of miR-149 levels and the underlying epigenetic regulatory mechanism in human GBM has not been studied. We used quantitative real-time polymerase chain reaction to investigate the levels of miR-149 in GBM tissues, their matched adjacent normal tissues, and glioblastoma U87MG cell line. Using bisulfite genomic sequencing technology, DNA methylation status of upstream region of miR-149 was evaluated in study population groups and the U87MG cell line. After treatment of cells with 5-aza-2′-deoxycitidine (5-aza-dC), the DNA methylation status, gene expression, and target protein levels of miR-149 were investigated. Our studies revealed that methylation and expression levels of miR-149 were significantly increased and decreased, respectively in GBM patients relative to the adjacent normal tissues (P < 0.01). MiR-149 suppressed the expression of AKT1 and cyclin D1 and reduced the proliferative activities of the U87MG cell line. Treatment of U87MG cells with 5-aza-dC reversed the hypermethylation status of miR-149, enhanced the expression of its gene, and decreased target mRNA and proteins levels (P < 0.01). These findings suggest that the methylation mechanism is associated with decreased expression levels of miR-149, which may in turn lead to the increased levels of its oncogenic target proteins.