Abstract
BackgroundNuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemo-sensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. However, the significance of RelB in colorectal cancer (CRC) remains unclear. Here, we systematically explored the functions of the alternative NF-κB subunit RelB in colon cancer cells and its underlying mechanism.MethodsStably transfected RelB-shRNA DLD-1 cells were established using Lipofectamine 2000. NF-κB DNA-binding capability was quantified using an ELISA-based NF-κB activity assay. Cell growth was monitored by an x-Celligence system. Cell proliferation was analyzed by a CCK-8 and a Brdu proliferation assay. Response to 5-FU was assessed by an x-Celligence system. Cell apoptosis and cell cycle was detected using flow cytometry analyses. Cell migration and invasion abilities were detected by an x-Celligence system, Transwell inserts, and wound-healing assays. RelB expression and its clinical significance were analyzed using the CRC tissue microarray. The expression of NF-κB signaling subunits, AKT/mTOR signaling molecules, cell cycle related proteins, MMP2, MMP9, and Integrin β-1 were measured by Western blotting analyses.ResultsThe RelB-silencing inhibited cell growth of DLD-1 cells. The RelB-silencing exerted the anti-proliferative by downregulation of AKT/mTOR signaling. The RelB-silencing caused G0–G1 cell cycle arrested likely due to decreasing the expression of Cyclin D1 and CDK4, concomitant with increased expression of p27Kip1. The RelB-silencing enhanced cytotoxic effect of 5-FU and induced cell accumulation in S-phase. The RelB-silencing impaired the migration and invasion potential of DLD-1 cells, which was related to downregulation of MMP2, MMP9, and Integrin β-1. Importantly, the RelB expression was correlated with depth of tumor invasion, lymph node metastasis, metastasis stage, and pTNM stage. High-RelB expression was significantly correlated with poor overall survival in CRC patients.ConclusionOur studies here provided evidence that RelB plays an oncogenic role and conveys chemo-resistance to 5-FU. RelB can be considered as an independent indicator of prognosis in CRC.
Highlights
Nuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemosensitivity
Introduction of RelB‐short hairpin RNA (shRNA) into DLD‐1 colon cancer cells The endogenous expression of all nuclear transcription factor kappa B (NF-κB) subunits in the whole-cell extracts was examined by Western blotting analysis in the three colon cancer cell lines including DLD1, HT-29, and Caco-2
The DLD-1 cells were transfected with plasmid carrying either RelB-shRNA or control-shRNA respectively
Summary
Nuclear transcription factor kappa B (NF-κB) subunits exhibit crucial roles in tumorigenesis and chemosensitivity. Recent studies suggest that RelB, the key subunit of the alternative NF-κB pathway, plays a critical role in the progression of diverse human malignancies. Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide, and is a multistep genetic disorder. The classical NF-κB pathway involves activation of the IκB kinase (IKK) complex (composed of IKKα, IKKβ, and IKKγ subunits), leading to phosphorylation of IκB proteins. This pathway usually regulates the nuclear translocation activity of p50/RelA and p50/cRel heterodimers. The NF-κB inducing kinase (NIK) activates IKKα, leading to the phosphorylation and proteasome-mediated partial degradation of p100 to generate p52, resulting in the formation of RelB/p52 complexes. The classical NF-κB activity has been implicated in the chemo-resistance and proteasome inhibition targeting NF-κB in CRC [14]
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