The Function And Mechanism Of The Nuclear Transcription Factor RelB In The Tumorigenesis Of Colorectal Cancer

Objective To investigate the role of the nuclear transcription factor RelB in the tumorigenesis of colorectal cancer(CRC)and its mechanism.Methods Establish a stable transfection of RelB-siRNA and ctrl-siRNA in DLD-1.The cell growth rates,cell migration and invasion assay were detected by a real-time x-Celligence system.Cell proliferation was detected by a CCK-8 assay and a Brdu assay.Cell cycle assay and cell apoptosis assay were analyzed by flow cytometry.Transwell chambers were used to examine the role of RelB on the migration and invasion abilities of the two established cell lines.The wound-healing assay was also carried out to evaluate the migration ability of DLD-1 cells.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 p-1 were measured by Western blotting analyses.Results The cellular growth was continuously monitored by a real-time x-Celligence system.The DLD-1-siRelB cells grew much slower than the DLD-1-sictrl cells(P<0.05).In CCK-8 and Brdu assay,the OD450 value was markedly decreased in the DLD-1-siRelB cells than the DLD-1-sictrl cells(P＜0.05 and P＜0.001).The distributions of G0-G1,S,and G2-M phase in the DLD-1-sictrl cells were 41.69±0.41%,27.65±0.06%,and 31.15±0.47%,while those in the DLD-1-siRelB cells were 57.65±0.36%,28.89±0.71%,and 13.16±1.30%,respectively.Cell cycle progression of the DLD-1-siRelB cells was notably arrested in the G0-G1 phase(P<0.001).In the clone formation assay,the numbers of clones formed by DLD-1-siRelB cells was significantly less than that of the control group.Western blotting assay was performed to examine the important molecules in the signaling.The total AKT stayed unchanged.The expression of phosphor-AKT(Thr308)and phosphor-AKT(Ser473)in the DLD-1-siRelB cells were evidently reduced compared to that of the DLD-1-sictrl cells.Phosphate and tension homology deleted on chromosome ten(PTEN),a negative regulator of the AKT signaling,Was induced in the DLD-1-siRelB cells.The phosphorylation of mTOR at Ser2448 and phosphorylation of p70S6K at Thr389 were deduced in the DLD-1-siRelB cells.Cyclin D1 and CDK4 were clearly decreased while p27Kipl was increased in the DLD-1-siRelB cells.The cell migration and invasion were continuously mouitored by x-Celligence system.The DLD-1-siRelB cells migrated(invaded)dramatically slower than the DLD-1-sictrl cells(P＜0.05).Transwell chambers were also used to examine the role of RelB on the migration and invasion abilities of the DLD-1 cells.The number of migrated DLD-1-siRelB cells was less than that of the DLD-1-sictrl cells(P<0.01).Consistently,the number of invaded(Transwell chambers pre-coated with Matrigel)DLD-1-siRelB cells was significantly lesser than that of the DLD-1-sictrl cells.In the wound healing assay,the DLD-1-siRelB cells migrated from the border towards the scratch center slower than the DLD-1-sictrl cells.Western blotting assay showed that the expression of MMP2,MMP9 and Integrin β1 in the DLD-1-siRelB was clearly decreased compared with that of the DLD-1-sictrl cells.Consistently,the mRNA expression of MMP2,MMP9 and Integrin β1 was decreased in the DLD-1-siRelB cells compared with that in the DLD-1-sictrl cells.Both the DLD-1-sictrl and DLD-1-siRelB cells were treated with different concentrations of 5-FUfor 48 h.The cell growth rate of the DLD-1-sictrl and DLD-1-siRelB cells upon treated with 5-FU was decreased in a dose-dependent manner.The IC50 of 5-FU for the DLD-1-siRelB cells was significantly lower compared with that of the control cells(P<0.001).Expose to 5-FU for 48 h,the distributions of G0-G1,S,and G2-M phase in the DLD-1-sictrl cells were 44.43±0.44%,30.31±0.70%,and 25.26±0.30%,while those in the DLD-1-siRelB cells were 56.35±1.04%,42.94±0.70%,and 0.71±0.40%.The induction of the cell arrest in the S-phase was significant in the DLD-1-siRelB cells upon treated with 5-FU compared with that of the control cells(14.05%vs.2.66%,p<0.001).The expression of Cyclin D1 was slightly decreased.To assess the putative clinical significance of RelB expression in CRC patients,RelB expression was identified in CRC tissues by IHC staining.The expression of RelB was positively correlated with depth of tumor invasion(P＝0.018),lymph node metastasis(P<0.001),metastasis stage(P-0.031),and pTNM stage(P=0.001).The OS in patients with high expression of RelB was significantly shorter than that in patients with low expression of RelB(P=0.006).High RelB expression was independent factor for shorter OS in CRC patients.Conclusions 1.RelB-silencing inhibited cell growth of DLD-1 cells due to the anti-proliferative by dovnregulation of AKT/mTOR signaling and G0-G1 cell cycle arrested.2.The RelB-silencing impaired the migration and invasion potential of DLD-1 cells,which was related to downregulation of MMP2,MMP9,and Integrin β-1.3.The RelB-silencing enhanced cytotoxic effect of 5-FU and induced cell accumulation in S-phase.4.RelB can be considered as an independent indicator of prognosis in CRC.