MiR-595 Suppresses the Cellular Uptake and Cytotoxic Effects of Methotrexate by Targeting SLC19A1 in CEM/C1 Cells.

Abstract

The human solute carrier family 19 member 1 (SLC19A1) is the gene coding for reduced folate carrier 1 (RFC1). In our previous work, we showed that the miR-595-related polymorphism, rs1051296G>T, which was located in the 3'-untranslated region (3'-UTR) of SLC19A1, was associated with high methotrexate (MTX) plasma concentrations in patients with paediatric acute lymphoblastic leukaemia (ALL). This study aimed to investigate the role of miR-595 in the regulation of SLC19A1 expression and its effects on the cellular uptake and cytotoxicity of MTX in ALL CEM/C1 cells. Luciferase reporter assay was performed to validate SLC19A1 as a miR-595 target. RFC1 protein expression was determined via Western blotting. Intracellular MTX concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Cell viability and apoptosis were assessed using Cell Counting Kit-8 (CCK-8) assay and flow cytometer, respectively. Compared to the negative control, miR-595 mimics induced a significant decrease in the relative luciferase activity by binding to the 3'-UTR of SLC19A1 harbouring the rs1051296 T allele (p<0.01). Treatment of CEM/C1 cells with miR-595 mimics substantially reduced RFC1 protein expression, intracellular MTX levels, MTX-induced cytotoxicity and apoptosis rates compared to those of negative control. However, opposite results were observed in cells transfected with a miR-595 inhibitor. These findings suggested that miR-595 acts as a phenotypic regulator of MTX sensitivity in CEM/C1 cells by targeting SLC19A1. This study helped us to understand the mechanisms underlying the variable MTX responses observed in patients with ALL.