Target inhibition on GSK-3β by miR-9 to modulate proliferation and apoptosis of bladder cancer cells

Abstract

Objective: Glycogen synthase kinase-3β (GSK-3β) can negatively regulate Wnt/β-catenin signaling pathway via degrading β-catenin protein, and plays suppressing roles in various tumors. Its role in bladder cancer pathogenesis is still unclear. In bladder cancer tissues, expression of microRNA-9 (miR-9) is significantly elevated. This study investigated the effect of miR-9 in modulating GSK-3β expression, Wnt/β-catenin pathway activity, and proliferation or apoptosis of bladder cancer cells.

Patients and methods: Dual luciferase reporter gene assay confirmed targeted regulation between miR-9 and GSK-3β. Bladder cancer tissues were collected to measure expression of miR-9, GSK-3β mRNA using adjacent tissues as the control. Expression of miR-9 and GSK-3β was also measured in HBEC, RT4 and TCCSUP cells. Cultured RT4 and TCCSUP cells were transfected with miR-9 inhibitor or pSicoR-GSK-3β. The expression of miR-9, GSK-3β and β-catenin was compared, followed by using flow cytometry assay for cell apoptosis and EdU staining for cell proliferation.

Results: Comparing to adjacent tissues, bladder cancer tissues illustrated significantly elevated miR-9 expression and lower GSK-3β mRNA. Bioinformatics analysis revealed complementary binding sites between miR-9 and 3'-UTR of GSK-3β mRNA, indicating targeted regulation between miR-9 and GSK-3β. Comparing to HBEC cells, RT4 and TCCSUP cells had significantly elevated miR-9 expression and lower GSK-3β expression, with enhanced proliferation. Transfection of miR-9 inhibitor or pSicoR-GSK-3β significantly elevated GSK-3β expression and suppressed β-catenin expression, promoted cell apoptosis and inhibited proliferation.

Conclusions: MiR-9 up-regulation plays a role in suppressing GSK-3β expression and facilitating bladder cancer pathogenesis. Inhibition of miR-9 could potentiate GSK-3β expression, suppress proliferation of bladder cancer, and facilitate apoptosis.