We first examined whether individual knockdown of these 7 genes can exert an effect on cell viability. but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the crucial role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis. retinoic acid Introduction Neuroblastoma is one of the most common solid cancers of child years. High-risk neuroblastoma is one of the leading causes of cancer-related deaths in child years,1,2 because only a few high-risk neuroblastoma patients become long-term survivors with currently available therapeutic agents for treating neuroblastoma. Differentiation therapy was developed based on the knowledge that neuroblastoma arises from the neural crest cell precursors that fail to total the differentiation process.2,3 It is an approach to induce malignant cells to differentiate into mature cells and thereby leading to tumor growth arrest.2,4-6 Currently, the differentiation agent 13-< 0.05, comparing to the negative control oligo at the corresponding time or dose points. (CCD), Effect of miR-449a mimic and precursor mimic on neurite outgrowth in Banoxantrone D12 BE(2)-C cells. Cells were transfected with Pfdn1 25?nM control oligo, miR-449a mimic or Banoxantrone D12 precursor mimic in triplicates, with neurite lengths measured as above after 4 d. Shown are the representative cell images (C) analyzed to define neurites (pink) Banoxantrone D12 and cell body areas (yellow), and the quantification of neurite length (D). *, < 0.05, comparing to control. (ECF), Effect of miR-449a mimic on neurite outgrowth in multiple neuroblastoma cell lines. Cells were transfected with 25?nM miR-449a mimic or control oligo in triplicates. Neurite lengths were measured as above. (E) Representative cell images analyzed to define neurites (pink) and cell body areas (yellow) after 4 d of transfection. (F) Quantification of neurite lengths. *, < 0.05, comparing to control. G, Effect of miR-449a overexpression around the protein expression levels of cell differentiation markers III-tubulin, NSE and Space43 with calnexin protein levels used as a loading control. Cells were transfected with 25?nM miR-449a mimic or control oligo, and protein levels were determined by Western blot after 4 d. (H) Endogenous expression levels of miR-449a in undifferentiated and differentiated BE(2)-C cells. BE(2)-C cells were treated with 10 M of RA or the carrier DMSO (Control) for 5 d to induce cell differentiation. RNA was then isolated, and expression of miR-449a in cells were measured by qPCR with levels of 18s rRNA as a loading control. *, < 0.05, comparing to control. Next we were interested in examining whether the endogenous expression of miR-449a is regulated during neuroblastoma cell differentiation. We measured the endogenous miR-449a levels in BE(2)-C cells that were induced into differentiation by RA (Fig.?S2). As shown in Figure?1H, the endogenous expression of miR-449a in differentiated BE(2)-C cells is significantly increased comparing to the undifferentiated (Control) cells. These results strongly suggest that the endogenous expression of miR-449a is suppressed in undifferentiated neuroblastoma cells. miR-449a overexpression reduces cell proliferation and induces apoptosis in neuroblastoma cells To further characterize the potential tumor suppressive function of miR-449a in neuroblastoma, we examined whether the induced cell differentiation by miR-449a is coupled with reduced neuroblastoma cell survival. As shown in Figure?2A, miR-449a mimic decreases cell viability in a dose-dependent manner in all the examined neuroblastoma cell lines. We further investigated the mechanisms underlying the reduced cell survival induced by miR-449a. As shown in Figure?2B, miR-449a overexpression significantly decreases DNA synthesis in the cells, as measured by the reduced bromodeoxyuridine (BrdU) incorporation into cell DNA,.