MicroRNA-28-5p regulates glioma cell proliferation, invasion and migration by targeting SphK1.

Abstract

MicroRNAs (miRNAs) are a conserved class of endogenous and short non-coding RNAs that post-transcriptionally regulate the expression of genes involved in diverse cellular processes. MiR-28-5p has been reported to be associated with several cancers, including human glioma. However, the roles of miR-28-5p in glioma development are poorly understood. Sixteen human glioma tissues and paired adjacent normal tissues were acquired through the Gansu Provincial Hospital. We performed quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) to detect the miR-28-5p expression between 16 paired adjacent normal and glioma tissues, as well as the miR-28-5p expression between normal human astrocytes cells and five glioma cell lines. To examine the functional roles of the downregulated miR-28-5p in glioma, cell viability and colony formation assays were performed for the analysis of cell growth. We overexpressed miR-28-5p by transient transfection of miRNAs mimics and performed the transwell Matrigel invasion assay and transwell migration (without Matrigel) assay. To investigate the roles of miR-28-5p in SphK1 expression, Western blot and Real Time-Polymerase Chain Reaction assays were performed. In this work, we demonstrated that miR-28-5p is downregulated in glioma tissues compared to the adjacent normal tissues. Functional studies showed that miR-28-5p overexpression inhibited the cell viability, colony formation and proliferation; meanwhile, it induced the cell apoptosis. The transwell invasion assay indicated that miR-28-5p blocked the invasion and migration of glioma cells. SphK1 (Sphingosine kinase 1 antibody) is predicted as a targeted candidate of miR-28-5p. Then, the Luciferase reporter assay, Western blot and Real Time-Polymerase Chain Reaction (PCR) validated that miR-28-5p negatively regulated SphK1 expression by directly targeting its 3'untranslated regions (3'UTR) in U87 cells. Furthermore, rescue assay suggested that overexpression of SphK1 without its 3'UTR could prevent the miR-28-5p from inducing the inhibition of glioma tumor cells. Our findings showed that miR-28-5p could suppress the growth, invasion and migration of glioma cells by suppressing the SphK1 expression. The results demonstrated that miR-28-5p might serve as an important potential therapeutic target for glioma.