title: MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway.
abstract: Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.
authors.affiliation: Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, York House, York Street, Dublin 2, Ireland.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lynch
authors.forename: Joanna
authors.initials: J
authors.lastname: Fay
authors.forename: Maria
authors.initials: M
authors.lastname: Meehan
authors.forename: Kenneth
authors.initials: K
authors.lastname: Bryan
authors.forename: Karen M
authors.initials: KM
authors.lastname: Watters
authors.forename: Derek M
authors.initials: DM
authors.lastname: Murphy
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Stallings
mesh_terms: D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics; D018450:Disease Progression; D015536:Down-Regulation; D006023:Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism; D000071447:N-Myc Proto-Oncogene Protein; D009361:Neoplasm Invasiveness; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D016212:Transforming Growth Factor beta / Q000235:genetics* / Q000378:metabolism*; D015854:Up-Regulation; D054460:rho-Associated Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D006023:Glycoproteins; C015246:LRG1 protein, human; C556347:MIRN335 microRNA, human; C459801:MYCN protein, human; D035683:MicroRNAs; D018994:Myosin Light Chains; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; C515851:ROCK1 protein, human; D054460:rho-Associated Kinases; C535150:MAPK1 protein, human; D019950:Mitogen-Activated Protein Kinase 1