miR-331-3p regulates expression of neuropilin-2 in glioblastoma

Michael R. Epis, Keith M. Giles, Patrick A. Candy, Rebecca J. Webster, Peter J. Leedman

Aberrant expression of microRNAs (miRNAs), a class of small non-coding regulatory RNAs, has been implicated in the development and progression of high-grade gliomas. However, the precise mechanistic role of many miRNAs in this disease remains unclear. Here, we investigate the functional role of miR-331-3p in glioblastoma multiforme (GBM). We found that miR-331-3p expression in GBM cell lines is significantly lower than in normal brain, and that transient overexpression of miR-331-3p inhibits GBM cell line proliferation and clonogenic growth, suggesting a possible tumor suppressor role for miR-331-3p in this system. Bioinformatics analysis identified neuropilin-2 (NRP-2) as a putative target of miR-331-3p. Using transfection studies, we validated NRP-2 mRNA as a target of miR-331-3p in GBM cell lines, and show that NRP-2 expression is regulated by miR-331-3p. RNA interference (RNAi) to inhibit NRP-2 expression in vitro decreased the growth and clonogenic growth of GBM cell lines, providing further support for an oncogenic role for NRP-2 in high-grade gliomas. We also show that miR-331-3p inhibits GBM cell migration, an effect due in part to reduced NRP-2 expression. Finally, we identified a significant inverse correlation between miR-331-3p and NRP-2 expression in The Cancer Genome Atlas GBM cohort of 491 patients. Together, our results suggest that a loss of miR-331-3p expression contributes to GBM development and progression, at least in part via upregulating NRP-2 expression and increasing cell proliferation and clonogenic growth.