MiR-505 Downregulates 6-Phosphofructo-2-Kinase/ Fructose-2,6-Biphosphatase 4 to Promote Cell Death in Glioblastoma

Abstract

Aim: The glycolytic enzyme, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4), mediates shifts in glycolytic flux and is important for glioblastoma cell survival. This study aimed to identify micro-RNAs that alter PFKFB4 expression to regulate glioblastoma cell survival. Methods: Western blot analyses, luciferase reporter assays, and public database analyses were used to predict and validate the regulation of PFKFB4 mRNA expression by miR-505 in glioblastoma. Cell growth and apoptosis assays were performed to determine the effects of miR-505 on the growth and survival of primary glioblastoma stem-like cells (GSCs) and established glioblastoma cell lines. In addition, the correlations between patient survival and the expression of miR-505 and PFKFB4 mRNA in glioblastoma specimens were examined. Results: Using micro-RNA target prediction programs, a miR-505 binding site in the 3'-UTR of the PFKFB4 mRNA transcript was identified, and query of a public CLIP-Seq database indicated that PFKFB4 binds this site in living cells. It was found that fusion of the PFKFB4 3'-UTR to luciferase conferred regulation of luciferase activity by PFKFB4. In addition, Western blots revealed that miR-505 significantly decreased PFKFB4 protein expression in established glioblastoma cell lines and primary GSCs. Enforced PFKFB4 overexpression increased the growth of primary GSCs and established glioblastoma multiforme cell lines, and miR-505 antagonized this effect. By downregulating PFKFB4, miR-505 increased production of reactive oxygen species, thereby repressing glioblastoma cell growth and promoting glioblastoma cell death. Importantly, patient survival was positively correlated with miR-505 expression and negatively correlated with PFKFB4 mRNA expression in primary glioblastoma specimens. Conclusion: It was showed that miR-505 downregulates PFKFB4 expression in glioblastoma, thereby decreasing glioblastoma cell survival. Targeting PFKFB4 via miR-505 may represent a promising therapeutic approach in glioblastoma.