Deregulation of focal adhesion pathway mediated by miR-659-3p is implicated in bone marrow infiltration of stage M neuroblastoma patients

Abstract

To get insights on the metastatic process of human neuroblastoma (NB), the miRNA expression profile of bone marrow (BM)-infiltrating cells has been determined and compared to that of primary tumors. Twenty-two BM-infiltrating cells, 22 primary tumors, and 4 paired samples from patients with metastatic NB aged > 12 months were analyzed for the expression of 670 miRNAs by stem-loop RT-qPCR. The miRNAs whose expression was significantly different were subjected to selection criteria, and 20 selected miRNAs were tested in 10 additional BM-infiltrating cells and primary tumors. Among the miRNAs confirmed to be differentially expressed, miR-659-3p was further analyzed. Transfection of miR- 659-3p mimic and inhibitor demonstrated the specific suppression and over-expression, respectively, of the miR-659-3p target gene CNOT1, a regulator of transcription of genes containing AU-rich element (ARE) sequence. Among the ARE-containing genes, miR-659-3p mimic and inhibitor specifically modified the expression of AKT3, BCL2, CYR61 and THSB2, belonging to the focal adhesion pathway. Most importantly, in BMinfiltrating cells CNOT1 expression was significantly higher, and that of AKT3, BCL2, THSB2 and CYR61 was significantly lower than in primary tumors. Thus, our study suggests a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in the human NB metastatic process.