Evaluation of angiogenic capacity of human adenocarcinoma cell line knockout for NF-ĸΒ1 protein

Abstract

INTRODUCTION: Renal cell carcinoma (RCC) is the most common adult renal epithelial cancer. The most frequent subtype of RCC is clear cell (ccRCC). Most of ccRCC patients have a mutation in the Von Hippel-Lindau (VHL) tumor suppressor gene. The VHL gene encodes a protein, the VHL, which can up-regulate a series of intracellular proteins, including the hypoxia inducible factor (HIF). The transcription factor NF-кB is increased in the ccRCC. OBJECTIVES: To evaluate the impact of the NF-кB1 gene knockout on the VEGF and IL6 expression in the human RCC cells under normoxia and hypoxia. MATERIALS AND METHODS: The CRISPR/Cas-9 technique was used to obtain 786-0 cells knockout for the NF-кB1 protein. Western Blot assay was used to selected the clones. A hypoxia-inducing humid chamber was used and its effectiveness was validated its effectiveness was certified by the analysis of HIF-2α expression levels. The quantification of VEGF and IL-6 levels was measured using Real Time-PCR and MILLIPLEX assay. DISCUSSION AND RESULTS: The VEGF gene expression in the clones was significantly lower than that presented by the control both in normoxia (786-0-sg1 99.68±0.09%, 786-0-sg2 78.55±0.85%, 786-0-sg3 91.70±0.87%) and in hypoxia (786-0-sg1 98.30±1.49%, 786-0-sg2 75.21±4.14%, 786-0-sg3 98.44±0.18%). The expression of IL-6 gene was also significant lower in normoxia (786-0-sg1 49.03±0.80%, 786-0-sg2 76.59±12.43%, 786-0-sg3 66.98±10.89%) and in hypoxia (786-0-sg1 95.85±0.36%, 786-0-sg2 96.45±0.49%, 786-0-sg3 91.08±1.42%). The MILLIPLEX results show that there was a significant reduction of both VEGF and IL-6 in the culture medium of cells knocked out in normoxia and hypoxia compared to control group. CONCLUSION: Suppression of p50 expression in the clones resulted in the reduction of VEGF and IL6 in both conditions. The reduction in the IL-6 relative expression hypoxia/normoxia demonstrates a change in cellular responsiveness to decreased levels of oxygen.