Abstract 781: The tumor suppressive microRNA-15 family targets MYCN in neuroblastoma

Abstract

Abstract Background: Neuroblastoma is the most common extracranial solid malignancy in children. Amplification/stabilization of the MYCN oncogene defines a group of neuroblastoma with a high risk of recurrence. Despite current aggressive treatment regimens, the prognosis for high-risk neuroblastoma patients remains poor with survival less than 40%. Most of the affected children ultimately die of the disease, which highlights the urgent need for novel therapeutic approaches. MicroRNAs (miRNAs) are small non-coding RNAs with gene expression regulatory functions whose expression is dysregulated in all types of human cancers, including neuroblastoma. However, the role of miRNAs in the regulation of MYCN and the development of drug resistance is currently unknown. Hypothesis: We hypothesize that miRNA-15 family act as a tumor suppressor in neuroblastoma. Methods: Neuroblastoma patient tumors were expanded in vivo. miRNAs were profiled by Nanostring and validated by qRT-PCR using TaqMan assays. miRNAs or their inhibitors were overexpressed by lipid reagent mediated transfection or lentiviral infection. Western blotting and qRT-PCR assays detected MYCN expression. Luciferase reporter and Ago-2 immunoprecipitation assays were performed to test miRNA-15 directly targeting of MYCN through interaction with 3’UTR of MYCN mRNA. Cell proliferation assay was carried out through MTT; cell migration by wound-healing; whereas invasion by examining the ability of cells to pass through a Matrigel-coated membrane matrix in neuroblastoma cells. Tumor growth was measured in neuroblastoma xenografts treated with neuroblastoma cells alone or with miRNA-15. MYCN (mRNA and protein), and miRNA-15 levers were assessed in xenograft tumors. Student’s t-test was used to evaluate the differences between treatment groups. All statistical tests were two-sided. Results: Based on the MYCN expression, neuroblastoma patient-derived xenografts (PDX) were divided into MYCN higher and lower expression. miRNA-15 family such as miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p were downregulated in PDX with higher MYCN expression. By luciferase and Ago-IP assays, we found miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p can directly bind with 3’UTR region of the MYCN mRNA. R2 database involving 105 neuroblastoma patients show an inverse correlation between MYCN mRNA and miRNA-15 host gene. The forced expression of the miRNA-15a-5p, miRNA-15b-5p, and miRNA-16-5p in neuroblastoma cells resulted in a significant reduction in the levels of MYCN and suppressed cell proliferation, migration, and invasion. Conversely, the inhibition of miRNA-15 family expression dramatically accelerated MYCN expression. Injection of neuroblastoma cells with miRNAs inhibits the tumor formation in neuroblastoma xenografts. Conclusion: These data suggest that miRNA-15 family may function as a tumor suppressor by targeting MYCN in neuroblastoma. Citation Format: Kishore B. Challagundla, Patrick Reynolds, Santhi Gorantla, Larisa Poluektova, Don Coulter, Surinder K. Batra, Srinivas Chava. The tumor suppressive microRNA-15 family targets MYCN in neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 781.