ABSTRACT: Presence of BM-infiltrating neuroblastoma (NB) cells occurs in about 50% of patients affected by this peculiar pediatric cancer. Children aged more than 18 months at diagnosis with metastatic NB are high-risk patients with 25-30% overall survival at 5 years, despite multimodal aggressive therapy. Little is known about the metastatic process in NB patients. Hereby, we focused on miRNA expression profiles of BM-infiltrating as compared to those of primary NB tumors. For this purpose, we analyzed 22 BM-infiltrating NB cells, 22 primary NB tumors, and 4 paired BM-infiltrating and primary tumor specimens, all from patients with metastatic disease and aged > 18 months at diagnosis. Statistical analysis indicated that in human BM-infiltrating cells miR-659-3p was down-modulated as compared to primary tumors, in all sets of samples. Functional studies in HTLA and SH-SY5Y NB cell lines by miR-659-3p mimic and inhibitors demonstrated that miR-659-3p regulates the expression of the transcription factor CNOT1, that in turn regulates the expression of AU-rich element (ARE)-containing target genes AKT3, BCL2, THSB2 and CYR61, all belonging to the focal adhesion pathway. Indeed, CNOT1 expression was found significantly higher, whereas that of the ARE-containing target genes was significantly lower in BM-infiltrating cells than in primary tumors. Our findings about a role of the focal adhesion pathway, regulated by miR-659-3p through CNOT1, in NB metastatic process are intriguing for the development of new therapeutic strategies aimed to interrupt the metastatic process. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results. HTLA-230 (kindly donated by Dr E. Bogenman, Los Angeles, USA) and SH-SY5Y (purchased from Banca Biologica and Cell Factory, Genoa, Italy) NB cell lines were cultured in RPMI 1640 medium supplemented with 10% heat inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin and 2 mM glutamine (Sigma-Aldrich, Milan, Italy). The HTLA-230 cells present MYCN amplification and SH-SY5Y cells have normal MYCN status. Both cell lines were checked for MYCN amplification, morphology, proliferation rate and mycoplasma contamination, after thawing and within four passages in culture. To evaluate the effect of miR-659-3p up- and down-regulation on gene expression, 1x106 HTLA-230 and SH-SY5Y cells were transfected with specific miR-659-3p mirVana™ mimic and inhibitor, respectively (Ambion, Life Technologies, Carlsbad, CA, USA, catalog# MC and MH 11582). Samples transfected with mirVana™ miRNA Mimic Negative Control #1 and mirVana™ miRNA Inhibitor Negative Control #1 were used as reference conditions. Transfection was performed at 20 nM miRNA concentration in OptiMEM© medium (Sigma-Aldrich), using Lipofectamin RNAiMAX© (Life Technologies), according to manufacturer’s protocol. Cells were then cultured for 48 hours, checked for viability and processed for total mRNA and miRNA extraction, as described below. Transfection experiments were performed twice with similar results.
