MicroRNA-149 is associated with clinical outcome in human neuroblastoma and modulates cancer cell proliferation through Rap1 independent of MYCN amplification

Abstract

PurposeWe evaluated the clinical relevance of microRNA-149 (miR-149) in neuroblastoma (NB) and its functional roles in regulating NB proliferation in vitro. MethodsQRT-PCR was used to evaluate miR-149 expression in NB cell lines and primary NB tumors. Association between endogenous miR-149 expression in primary NB tumors and their host patients' clinicopathological factors and overall survival (OS) were statistically evaluated. In SH-SY5Y, an MYCN-non-amplified, and LAN5, an MYCN-amplified NB cell lines, miR-149 was either upregulated or downregulated by lentiviral transduction, to evaluate its effect on NB proliferation in vitro. Possible downstream target of miR-149, Ras-related protein 1 (Rap1), was evaluated by qRT-PCR and western blot in lentiviral-transduced NB cells. Moreover, Rap1 was either upregulated or downregulated in lentiviral-transduced NB cells to further evaluate its effect on miR-149-mediated NB proliferation in vitro. ResultsMiR-149 is markedly downregulated in both in vitro NB cell lines and in vivo NB primary tumors. Low miR-149 expression is predominantly associated with Stage 3 or 4 primary NB tumors, and poor OS among NB patients. In SH-SY5Y and LAN5 cells, lentivirus-induced miR-149 upregulation inhibited, whereas miR-149 downregulation promoted NB proliferation in vitro, despite MYCN-amplification status. Rap1 expression, at both mRNA and protein levels, was inversely associated with miR-149 in NB. In addition, Rap1 upregulation or downregulation reversely regulated miR-149-mediated NB proliferation in vitro. ConclusionMiR-149 is downregulated in NB and closely associated with NB patients' clinical outcome. MiR-149 also functionally modulates NB cell proliferation in vitro, possibly through inverse-regulation on Rap1.