Abstract B41: miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1

Abstract

Purpose: Medulloblastoma, a most common pediatric malignant brain tumor consists of four molecular subgroups viz. WNT, SHH, Group 3 and Group 4. MiR-148a is over-expressed in the WNT subgroup tumors, which have the lowest incidence of metastasis and excellent survival among all medulloblastomas. Therefore, the role of miR-148a in medulloblastoma biology was investigated. Experimental Design: MiR-148a was expressed either in a transient manner using synthetic mimic or in a stable doxycycline inducible manner using a lentiviral vector. Effect of miR-148a expression on the growth and malignant behavior of medulloblastoma cell lines was investigated. CpG Methylation status of region upstream to pri-miR-148a was determined by performing bisulphite sequencing of this region in medulloblastoma cell lines and tissues. Results: Expression of miR-148a to the levels comparable to that in the WNT subgroup tumors was found to inhibit proliferation, clonogenic potential and in vitro invasion potential of medulloblastoma cells. In vivo tumorigenicity of medulloblastoma cells Daoy, D425 and D283, as seen by either generating subcutaneous xenografts or orthotopic xenografts in immunodeficient mouse, was found to be reduced significantly upon miR-148a expression. NRP1, ROCK1 and DNMT1 were identified as direct targets of miR-148a with NRP1 being novel target. Restoration of NRP1 expression in medulloblastoma cells was found to rescue the reduction in the invasion potential and tumorigenicity brought about by miR-148a expression. NRP1expression in medulloblastomas was found to be associated with poor survival, with little or no expression in majority of the WNT tumors. This observation is consistent with high miR-148a expression and low incidence of metastasis and excellent survival of the WNT subgroup tumors. miR-148a is known to be down-regulated as a result of promoter hypermethylation in colon, lung, breast, head and neck carcinomas and melanoma. Bisulphite sequencing of the region upstream to pri-miR-148a revealed presence of CpG methylation in four medulloblastoma cell lines Daoy, D283, D341 and D425 and non WNT medulloblastoma tissues, while it was found to be un-methylated in WNT medulloblastomas. Conclusions: The tumor suppressive effect of miR-148a expression in medulloblastoma cells accompanied by the down-regulation of NRP1, ROCK1 and DNMT1 makes miR-148a an attractive therapeutic agent for the treatment of medulloblastomas. Preliminary findings provided by bisulphite sequencing suggest that under-expression of miR-148a may be as a result of CpG methylation of upstream region to pri-miR-148a sequence. Citation Format: Kedar Narsinha Yogi, Epari Sridhar, Naina Goel, Rakesh Jalali, Atul Goel, Aliasgar Moiyadi, Rahul Thorat, Pooja Panwalkar, Atul Khire, Archya Dasgupta, Prakash Shetty, Neelam Shirsat. miR-148a functions as tumor-suppressor microRNA in medulloblastoma cell lines by targeting Neuropilin1. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr B41.