Downregulation of miR-181b inhibits human colon cancer cell proliferation by targeting CYLD and inhibiting the NF-κB signaling pathway

Abstract

It has been reported that microRNA (miRNA/miR)-181b plays an important role in regulating cellular proliferation, invasion and apoptosis in various tumors. However, the role of miR-181b and its molecular mechanisms in colon cancer cells have not yet been elucidated. The present study thus aimed to investigate the mechanisms of miR-181b targeting cylindromatosis (CYLD) to regulate the nuclear factor-κB (NF-κB) signaling pathway, and to determine its role in colon cancer cell proliferation and apoptosis. For this purpose, miR-181b was overexpressed and silenced in the SW480 cell line. The cell proliferation and apoptotic rates were determined using a Cell Counting kit and colony formation assays, and Annexin V-FITC staining, respectively. The expression levels of proteins associated with the NF-κB signaling pathway and apoptosis were detected by western blot analysis. Furthermore, a dual luciferase assay was applied to confirm the interaction between miR-181b and CYLD. CYLD was also overexpressed and silenced in the SW480 cell line using a CYLD overexpression plasmid and siRNA technology, respectively. Transfected cells were used for subsequent experiments. In addition, a nude mouse model was established to measure tumor volume and weight. Immunohistochemistry and a TUNEL assay were performed to detect the Ki67 levels and the cell apoptotic rate, respectively. Compared with the control group, miR-181 silencing or CYLD overexpression significantly attenuated cell proliferation, invasion and migration, and notably increased the proportion of apoptotic cells. Furthermore, the expression levels of Bax and cleaved caspase-3 were markedly increased, whereas those of Bcl-2 were significantly decresaed (P<0.05). In addition, the protein expression levels of p-p65/p65 and p-IκBα/IκBα were significantly downregulated and upregulated, respectively (P<0.05). Consistent with the results obtained in vitro, in vivo experiments using a nude mouse model yielded similar findings. The aforementioned results indicated that miR-181b downregulation inhibited human colon cancer cell proliferation by targeting CYLD to attenuate the activity of the NF-κB signaling pathway.