Since the respective miRNA clusters are transcribed in polycistronic transcripts and organized in four different families with high sequence homology, our results might indicate a certain amount of interchangeability of the cluster-related miRNAs as miR-20a and miR-106a are part of the same miRNA-family like miR-17 and miR-106b (Fig. including prognosis, metastases and response to neoadjuvant therapy. Interestingly, higher expression levels of specific miRNAs were significantly associated with an adverse outcome of patients and were also higher in patients with systemic spread. We could furthermore show a direct correlation between the expression of cluster activators (MYC, E2F1-3), inhibitors (TP53), individual miRNAs, and pro-apoptotic targets (FAS, BIM). Our findings therefore underline a critical role of the miR-17-92 Rolapitant cluster and its two paraloga in OS biology with pathogenetic and prognostic impact. strong class=”kwd-title” Keywords: osteosarcoma, miR-17-92, miR-106a-363, miR-106b-25, FAS, BIM INTRODUCTION Osteosarcomas (OS) are the most common primary malignant tumors of bone generally affecting the metaphyses of long bones in children and adolescents [1]. Due to a high rate of systemic spread already at the time of diagnosis patients greatly benefit from (neo-) adjuvant polychemotherapy in addition to radical surgery and reach 10-year survival rates of up to 73% in case of good response to cytostatic regimens [2, 3]. However, a substantial group of patients with metastatic, recurrent and/or refractory disease still lacks effective treatment options underlining the urgent need for new therapeutic alternatives and targets. Furthermore, there are no established biomarkers in OS that could identify patients with particularly aggressive tumors and could therefore constitute a basis for a more individualized treatment stratification [4]. One reason for this phenomenon is the genetic heterogeneity and complexity that is characteristic for OS and which hampers the identification of initiating and/or sustaining oncogenetic drivers. Amongst the most commonly mutated and/or altered genes in OS, TP53 and MYC have been identified, both of which are known to be deregulated in a variety of malignant tumors [1, 4]. Besides conventional oncogenes and tumor suppressors, microRNAs (miRNA) have increasingly been recognized as regulators of gene expression that can acquire oncogenic potential. The miR-17-92 cluster, also named oncomir-I, and its two paraloga miR-106a-363 and miR-106b-25 were among the first families of those small RNA molecules that were found to be upregulated in several malignant tumors. Meanwhile, several Rolapitant cluster-related miRNAs were shown to accelerate tumor development, to induce angiogenesis, to prevent apoptosis, and, only recently, to crucially influence osteoblastic proliferation and differentiation [5-7]. All three clusters are part of elaborate regulatory networks and can influence the expression of various genes involved in cell cycle control, apoptosis and angiogenesis (Figure ?(Figure1).1). Interestingly, MYC is known to stimulate the expression of cluster-related miRNAs whereas TP53 seems to have an inhibitory effect [8, 9]. In a previous study we demonstrated the upregulation of several of the respective miRNAs in a panel of established OS cell lines (HOS58, U2-OS, Saos-2, MNNG/HOS, SJSA-1, and MG-63) which was meanwhile confirmed by an independent Nr4a3 group [10, 11]. Open in a separate window Figure 1 The miR-17-92 cluster and its two paraloga miR-106a-363 and miR-106b-25 are centered in a complex network of regulators (upper half) and targets (lower half) of which this scheme only shows a selection for a better overview [8, 9]. In this study, we assembled a series of well characterized pretherapeutic OS samples to validate our cell line results in tumor biopsies and to analyze if the expression of individual miRNAs correlated with clinico-pathological parameters including prognosis, metastatic disease and/or response to therapy. In a next step, we interrogated the expression of selected regulators (MYC, TP53, E2F1, E2F2, E2F3) and pro-apoptotic targets (FAS, BIM) of the miR-17-92 cluster and its two paraloga that have already been described to show an altered expression and potential pathogenetic impact in OS [1, 4, 12, 13]. By this means, we aimed to confirm the upregulation of cluster related miRNAs and to provide a deeper insight into the causes and consequences of cluster activation in human osteosarcoma. RESULTS Patients and samples characteristics All patients characteristics are presented in Table ?Table1.1. MicroRNA stability is known to efficiently enable expression analyses in a variety of tissue sources. In this study we extracted miRNAs from 75 formalin fixed paraffin embedded (FFPE) pretherapeutic osteosarcoma samples. The expression levels of all miRNAs were.2013;25(4):398C406. and pro-apoptotic targets (FAS, BIM). Our findings therefore underline a critical role of the miR-17-92 cluster and its two paraloga in OS biology with pathogenetic and prognostic impact. strong class=”kwd-title” Keywords: osteosarcoma, miR-17-92, miR-106a-363, miR-106b-25, FAS, BIM INTRODUCTION Osteosarcomas (OS) are the most common primary malignant tumors of bone generally affecting the metaphyses of long bones in children and adolescents [1]. Due to a high rate of systemic spread already at the time of diagnosis patients greatly benefit from (neo-) adjuvant polychemotherapy in addition to radical surgery and reach 10-year survival rates of up to 73% in case of good response to cytostatic regimens [2, 3]. However, a substantial group of patients with metastatic, recurrent and/or refractory disease still lacks effective treatment options underlining the urgent need for new therapeutic alternatives and targets. Furthermore, Rolapitant there are no established biomarkers in OS that could identify patients with particularly aggressive tumors and could therefore constitute a basis for a more individualized treatment stratification [4]. One reason for this phenomenon is the genetic heterogeneity and complexity that is characteristic for OS and which hampers the identification of initiating and/or sustaining oncogenetic drivers. Amongst the most commonly mutated and/or altered genes in OS, TP53 and MYC have been identified, both of which are known to be deregulated in a variety of malignant tumors [1, 4]. Besides conventional oncogenes and tumor suppressors, microRNAs (miRNA) have increasingly been recognized Rolapitant as regulators of gene expression that can acquire oncogenic potential. The miR-17-92 cluster, also named oncomir-I, and its two paraloga miR-106a-363 and miR-106b-25 were among the first families of those small RNA molecules that were found to be upregulated in several malignant tumors. Meanwhile, several cluster-related miRNAs were shown to accelerate tumor development, to induce angiogenesis, to prevent apoptosis, and, only recently, to crucially influence osteoblastic proliferation and differentiation [5-7]. All three clusters are part of elaborate regulatory networks and can influence the expression of various genes involved in cell cycle control, apoptosis and angiogenesis (Figure ?(Figure1).1). Interestingly, MYC is known to stimulate the expression of cluster-related miRNAs whereas TP53 seems to have an inhibitory effect [8, 9]. In a previous study we demonstrated the upregulation of several of the respective miRNAs in a panel of established OS cell lines (HOS58, U2-OS, Saos-2, MNNG/HOS, SJSA-1, and MG-63) which was meanwhile confirmed by an independent group [10, 11]. Open in a separate window Figure 1 The miR-17-92 cluster and its two paraloga miR-106a-363 and miR-106b-25 are centered in a complex network of regulators (upper half) and targets (lower half) of which this scheme only shows a selection for a better overview [8, 9]. In this study, we assembled a series of well characterized pretherapeutic OS samples to validate our cell line results in tumor biopsies and to analyze if the expression of individual miRNAs correlated with clinico-pathological parameters including prognosis, metastatic disease and/or response to therapy. In a next step, we interrogated the expression of selected regulators (MYC, TP53, E2F1, E2F2, E2F3) and pro-apoptotic targets (FAS, BIM) of the miR-17-92 cluster and its two paraloga that have already been described to show an altered expression and potential pathogenetic impact in OS [1, 4, 12, 13]. By this means, we aimed to confirm the upregulation of cluster related miRNAs and to provide a deeper insight into the causes and consequences of cluster activation in human osteosarcoma. RESULTS Patients and samples characteristics All patients characteristics are presented in Table ?Table1.1. MicroRNA stability is known to efficiently enable expression analyses in a variety of tissue sources. In this study we extracted miRNAs.