Background Neuroblastoma (NB) is the most common extracranial solid tumor in children. Changes in tumorigenicity were assayed by soft agar colony forming ability. N-myc binding to miR-375 promoter was assayed by chromatin-immunoprecipitation. Results Unsupervised hierarchical clustering of miRNA microarray data segregated neuroblastic and non-neuronal cell lines and showed that specific miRNAs define each phenotype. qRT-PCR validation confirmed that increased levels of miR-21 miR-221 and miR-335 are associated with the non-neuronal phenotype whereas increased levels of miR-124 and miR-375 are GF 109203X exclusive to neuroblastic cells. Downregulation of miR-335 in non-neuronal cells modulates expression levels of HAND1 and JAG1 known modulators of neuronal differentiation. Overexpression of miR-124 in stem cells induces terminal neuronal differentiation with reduced malignancy. Expression of miR-375 is exclusive for N-myc-expressing neuroblastic cells and is regulated by N-myc. Moreover miR-375 downregulates expression of the neuronal-specific RNA binding protein HuD. Conclusions Thus miRNAs define distinct NB cell phenotypes. Increased levels of miR-21 miR-221 and miR-335 characterize the non-neuronal non-malignant phenotype and miR-335 maintains the non-neuronal features possibly GF 109203X by blocking neuronal differentiation. miR-124 induces terminal neuronal differentiation with reduction in malignancy. Data suggest N-myc inhibits neuronal differentiation of neuroblastic cells possibly by upregulating miR-375 which in turn suppresses HuD. As tumor differentiation state is highly predictive of patient survival the involvement of these miRNAs with NB differentiation and tumorigenic state could be exploited in the development of novel therapeutic strategies for this enigmatic childhood cancer. proto-oncogene and cellular heterogeneity are two key factors that influence patient survival. The three basic cell types Rabbit Polyclonal to CHRM2. in NB tumors and derived cell lines differ in their morphological biochemical and tumorigenic properties – whereas N-type neuroblastic cells are mildly malignant and have neuronal characteristics S-type cells are non-tumorigenic with features of non-neuronal (glial melanocytic and smooth muscle) precursor cells. I-type cancer stem cells which can differentiate into either N or S cells express stem cell marker proteins and are highly tumorigenic [2-4]. Thus the three basic cell phenotypes represent distinct differentiation states of NB with distinct tumorigenic properties. All three cell types are present in tumors [4]. Clinically cellular heterogeneity is predictive of patient outcome – patients with stroma-poor tumors comprising undifferentiated neuroblasts are frequently fatal whereas stroma-rich tumors or those with differentiated ganglion cells show a better prognosis [5]. Therefore one GF 109203X approach GF 109203X to controlling the malignant potential of this tumor involves exploiting its unique differentiation capacity. MicroRNAs (miRNAs) GF 109203X are important regulators of gene expression and function and hence differentiation. A role for miRNAs in neuroblastoma has been extensively studied mainly focusing on their association with respect to N-amplification chromosomal imbalances GF 109203X prognosis and retinoic acid (RA)-induced differentiation as discussed in four reviews [6-9]. These studies have revealed that large scale chromosomal imbalances result in dysregulated miRNAs which have a functional role in neuroblastoma pathogenesis and tumorigenicity. MiRNAs associated with N-amplification such as miR-17-92 cluster members are shown to be associated with NB tumorigenicity. Also miRNAs associated with RA-induced differentiation of NB has been extensively studied as RA is used clinically in treating NB patients. These studies as reviewed by Stalling et al. indicate that miRNA and DNA methylation changes following RA-treatment play a critical role in NB differentiation [9]. miRNAs modulated upon RA-treatment are shown to regulate key genes involved in differentiation survival and tumorigenic properties of NB [9]. The present study is mainly focused on investigating the association of miRNAs with respect to the different cell phenotypes derived from NB and.
