The Role Of Nuclear Transcription Factor RelB In Non-small Cell Lung Cancer

Lung cancer is the leading cause of cancer-related death worldwide with high incidence rate.Non-small cell lung cancer and Small cell lung cancer are the two main histologicaltypes of lung cancer.NSCLC accounts for approximately 80%-85%of lung cancer diagnoses.Although therapeutic approaches have improved drastically in the last decades,the prognosis of lung cancer patients is still not optimal with overall survival no more than 15%.NSCLC is a highly heterogeneous disease.Several driven genes contribute to aberrant cell proliferation and apoptosis in NSCLC.Therefore,it is important to identify useful molecular makers to investigate a novel therapeutic strategy in patients with NSCLC.RelB is the main subunit of the non-canonical NF-κB pathway.RelB expression was mainly detected in the cytoplasm of cells as an inactive form.The signaling pathway is activated by NIK(NF-κB inducing kinase,NIK)inducing NF-κB2/p100 protein to p52,resulting in transcriptionally activated RelB/p52 hetero-dimers.Activated RelB participates in various biological processes and play critical roles in immune and inflammatory responses,tumorigenesis and lymphocytic development.In prostate cancer,RelB is highly expressed in androgen-independent prostate cancer cells,and is correlated with Gleason score.RelB induced cell growth and radiosensitivity.Our results revealed that RelB is involved in various biological processes in prostate cancer cells,including cell apoptosis,migration and invasion.However,the expression and the mechanism of the RelB involved in NSCLC remain unclear.To investigate the function of RelB in the tumorigenesis and development in NSCLC,tissues after surgical treatment and clinicopathological information were obtained.RelB expression was analyzed in the tumor and adjacent non-neoplastic adenocarcinoma tissues to identify the possibility of RelB as an independent prognostic predictor.Then,NSCLC cells line with RelB silencing was established using RNA interference technology.We examined systemically the biological significance and radiosensitivity of RelB in NSCLC cells line.Object:To investigate the correlation between RelB expression and clinicopathological features of NSCLC patients and to explore the possibility of RelB as an independent prognostic indicator of NSCLC patients.Methods:The expression of RelB in NSCLC tumor tissue and adjacent non-neoplastic tissues were examined by immunohistochemistry and q RT-PCR.Chi-square tests were used to analyze possible associations between qualitative clinicopathological variables and RelB expression.Kaplan-Meier analysis and a Cox regression model were employed to determine independent prognostic factors.Results:IHC results revealed that RelB expression was mainly detected in the cytoplasm of NSCLC cells,and could hardly be detected in the adjacent nonneoplastic tissue in patients.High RelB expression was detected in 53/83(51.8%)adenocarcinoma patients and 17/32(53.1%)squamous cell carcinoma with no significance difference.The results of RelB m RNA in 15 pairs tumor tissue and adjacent non-neoplastic tissues q RT-PCR revealed that the m RNA expression of RelB was increased in tumor tissue compared with adjacent non-neoplastic tissue in NSCLC patients(p=0.003).High RelB expression was significantly correlated with depth of tumor invasion(p<0.001),lymph node metastasis(p=0.017),distant metastases(p=0.004),and TNM stages(p<0.001)in patients with NSCLC.These were no significant difference RelB expression and gender,age,smoke,differentiation and histological types of lung cancer.Kaplan-Meier and log-rank analysis revealed that NSCLC patients with high RelB expression had significantly shorter overall survival than those with low RelB expression(χ~2=32.993,p<0.001).A Cox regression model revealed that differentiation(HR=2.608,p=0.004),depth of tumor invasion(HR=2.196,p=0.015),lymph node metastasis(HR=2.386,p=0.014),distant metastases(HR=2.726,p=0.009),TNM stage(HR=3.148,p<0.001)and high RelB expression(HR=6.942,p<0.001)were correlated with overall survival.Low differentiation(HR=2.522,p=0.008)and high RelB expression(HR=6.983,p<0.001)were independent prognostic indicators for patients with NSCLC.Conclusions:The expression of RelB was increased in tumor tissue compared with adjacent non-neoplastic tissue in NSCLC patients.High RelB expression indicates bad prognosis and provide an independent prognostic indicator for patients with NSCLC.Object:To establish a stable shRNA-RelB NSCLC cell line.To investigate the function and the mechanism of RelB silencing on the cell growth,proliferation,apoptosis,migration,invasion and radio-sensitivity of SPC-A1.Methods:1.A shRNA carrying sequence targeting the RelB gene was designed and synthesized.The shRNA-RelB was then subcloned into the p Silencer3.1-H1-neo plasmid after anneal,renaturation and phosphorylation.2.Then transfected E.coli TOP 10competent bacteria.Positive monoclonal bacteria was selected and sequenced.The recombinant plasmid p Silencer3.1-ps RelB and the scrambled control plasmid were then transfected into SPC-A1 cells using Lipofectamine2000.The selected monoclones were further expanded and examined for the RelB expression by RT-PCR and western blot.3.The growth of SPC-A1-siRelB and SPC-A1-sictrl cells was detected by a real-time x-Celligence system.Xenograft tumor model was established to analyze the influence of RelB silencing on the tumorigenesis of SPC-A1.IHC was used to identify the RelB expression of the xenograft tumor.4.Cell proliferation,apoptosis and cell cycle were detected using FACSCalibur?cytometer for the SPC-A1-siRelB and SPC-A1-sictrl cells.The influence of RelB silencing on AKT signaling pathway was detected by western blot.5.The migration ability of SPC-A1-siRelB and SPC-A1-sictrl cells was detected by a real-time x-Celligence system and scratch healing assay.Gelatin zymography experiment was performed to identify the function of RelB silencing on the invasion of SPC-A1.The influence of RelB silencing on integrinβ-1 was detected by western blot.6.SPC-A1-siRelB and SPC-A1-sictrl cells were culture for another 96 hours after receiving a single dose of 8 Gy per treatment.Cell apoptosis was analyzed by flow cytometry.The protein Bcl-xl and Bcl-2 expression were analyzed by western blot,respectively.Results:1.The recombinant shRNA-RelB plasmid is successfully constructed.2.A stable shRNA-RelB NSCLC cell line was established by by RT-PCR and western blot.RelB silencing does not affect other NF-κB subunits.3.The SPC-A1-siRelB cells grew much slower than that of the SPC-A1-sictrl cells by a real-time x-Celligence system.Xenograft tumor assay revealed that the average volume of formed subcutaneous tumors from either the SPC-A1-siRelB cells or SPC-A1-sictrl cells were(0.36±0.31)cm~3 and(0.89±0.37)cm~3,respectively.The average weight of formed subcutaneous tumors from the SPC-A1-siRelB cells and SPC-A1-sictrl cells was(0.74±0.26)g and(1.03±0.22)g,respectively.There were significantly statistical differences in the volume(p=0.003)and weight(p=0.046)of subcutaneous tumors from the two established cell lines.IHC assays showed that RelB expression could be detected in the tumors tissues injected with SPC-A1-sictrl cells.However,RelB expression was hardly detected in the tumor tissues injected with SPC-A1-siRelB cells.Taken together,these data indicated that RelB silencing in the SPC-A1 cells suppress the cell growth in vitro and in vivo.4.The SPC-A1-siRelB cells proliferated markedly slower than the SPC-A1-sictrl cells.No significant differences in the spontaneous apoptosis rate and cell cycle were found between the SPC-A1-siRelB cells and the SPC-A1-sictrl cells at different time points.The phosphorylated AKT protein expression(phosphoration site at both Ser 308 and Ser 473)of SPC-A1-siRelB cells was distinctly reduced than that of the SPC-A1-sictrl cells.5.The SPC-A1-siRelB cells migrated distinctly slower than that of the SPC-A1-sictrl cells by a real-time x-Celligence system.The SPC-A1-siRelB cells migrated from the edge towards the scratch center much slower than that of the SPC-A1-sictrl cells in the scratch assay.Gelatin zymography experiment revealed that the MMP-9 activity was inhibited by the RelB silencing.6.The SPC-A1-siRelB and the SPC-A1-sictrlcells were culture for another96 hours after subjected an ionizing irradiation at 8 Gy.Subsequent to radiation exposure,apoptosis was measured using the Annexin V/PI flow cytometry assay at 24 h,48 h,72 h,and 96 h.The SPC-A1-siRelB cells had a much higher apoptosis rate than that of the SPC-A1-sictrl cells in the Annexin V/PI flow cytometry assay.There was a statistical significant difference in the apoptosis rate between the two groups(p<0.05).The expression of Bcl-xl protein was decreased in the SPC-A1-siRelB cells compare to that of the SPC-A1-sictrl cells after radiation exposure of 8 Gy at 96 h.The expression level of Bcl-2 stayed unchanged in response to irradiation.Conclusions:1.SPC-A1-siRelB cells was established successfully.2.RelB silencing suppressed the cell growth,attributed to the inhibited proliferation regulated by the deduced AKT phosphorylation.3.RelB-silencing attenuated the migration and invasion abilities of SPC-A1 cells,and is likely related to downregulate integrinβ-1 expression.4.RelB-silencing in SPC-A1 cells enhanced the radio-sensitivity,likely attributed to the downregulation of the Bcl-xl expression.