ABSTRACT
The Gax gene has been implicated in a variety of cell-developmental and biological processes, and aberrant Gax expression is linked to many diseases. In this study, to provide important insights for Gax-based gene therapy in vein graft restenosis and its anti-restenotic mechanism, we used rabbit vascular smooth muscle cells (VSMCs) to investigate the effects of Gax overexpression on proliferation, migration, cell cycle, and apoptosis in a serum-stimulated culture. Rabbit VSMC lines that stably overexpressed Gax were established by transfection with recombinant adenoviral vector Ad5-Gax. The effect of Gax overexpression on in vitro serum-induced VSMCs proliferation, migration, cell cycle, and apoptosis was assessed by MTT, wound healing, and flow cytometry assays, respectively. To investigate the effect of Gax overexpression on PCNA and MMP-2 in serum-induced VSMCs, immunocytochemistry, RT-PCR, and gelatin zymography were performed. The results clearly showed that Gax overexpression decreases PCNA expression in serum-induced VSMCs. Gax overexpression also significantly inhibited cell proliferation by blocking entry into the S-phase of the cell cycle, promoted cell apoptosis, and reduced cell migration activity by downregulating MMP-2 release and activity. These findings indicate that Gax would be an optimal target gene for gene therapy to treat vein graft restenosis.
Subject(s)
Gene Expression , Homeodomain Proteins/genetics , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Adenoviridae/genetics , Animals , Apoptosis/genetics , Cell Cycle/genetics , Cell Movement/genetics , Cell Proliferation , Cells, Cultured , Genetic Vectors/genetics , Male , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Proliferating Cell Nuclear Antigen/genetics , Proliferating Cell Nuclear Antigen/metabolism , Rabbits , TransfectionABSTRACT
PURPOSE: MicroRNA-31 (miR-31) has different expression patterns in various human cancers. Especially in urothelial carcinoma of the bladder, it has been demonstrated to be decreased expression in the invasive tumors and homozygously deleted. However, its clinical significance in human bladder cancer has not yet been elucidated. Thus, the purpose of this study was to investigate the diagnostic and prognostic values of miR-31 in this disease. METHODS: Expression levels of miR-31 in 126 pairs of bladder cancer and adjacent normal tissues were detected by real-time quantitative RT-PCR assay. To determine its prognostic value, overall survival (OS) and progression-free survival (PFS) were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. RESULTS: MiR-31 expression in bladder cancer tissues was significantly lower than those in adjacent normal tissues (mean expression level: 2.1 ± 0.9 vs. 3.8 ± 1.2, P < 0.001). When categorized into low vs. high expression, low miR-31 expression was negatively associated with the tumor stage (P = 0.02), the status of recurrence (P = 0.01), progression (P = 0.01), and death (P = 0.006) of patients with bladder cancer. Moreover, low miR-31 expression clearly predicted poorer PFS (P = 0.001) and OS (P < 0.001). In the multivariate analysis, low miR-31 expression was an independent prognostic factor for both PFS (P = 0.01) and OS (P = 0.008). CONCLUSION: These findings show that miR-31 may contribute to the progression of bladder cancer and its downregulation may be independently associated with unfavorable PFS and OS, suggesting that miR-31 might be a promising marker for further risk stratification in the treatment of this cancer.