NF-?Bp65 Regulates Tumorigenicity And The Expression Of Stem Cell Genes In Nasopharyngeal Carcinoma Cells

Objective: Nasopharyngeal carcinoma(NPC)is a malignant neoplasm originating from nasopharyngeal epithelium.It is highly prevalent in Southeast Asia and southern China.It has the characteristics of high local recurrence rate and early distant metastasis.Nuclear transcription factor-kappa B(NF-kappa B)is a member of the Rel protein family.It exists widely in many tissues and cells and regulates the expression of many genes.NF-kappa B not only involves physiological processes such as immune response,cell growth,survival and development,but also plays an important role in the occurrence and development of autoimmune diseases,inflammation and malignant tumors.In this study,we intend to construct a stable NPC 5-8F cell line transfected with the expression vector of siRNA plasmid specific to NF-kappa Bp65,to explore the effect of silencing of NF-kappa Bp65 on the cloning ability and tumorigenicity of 5-8F cells,and to detect the stem cell-related molecules affected by silencing of NF-kappa Bp65,so as to provide experimental basis for targeted stem cell therapy of NPC.Methods:(1)NF-kappa Bp65 siRNA plasmid expression vector and negative plasmid vector were transfected into nasopharyngeal carcinoma 5-8F cells,respectively.The transfected cells were screened by G418 and expanded to obtain stable transfected monoclonal cell lines.Western blotting was used to identify NF-kappa Bp65 silenced 5-8F cell lines and negative control 5-8F cell lines.Experimental groups: blank control group,negative control group and NF-kappa Bp65 silenced group.(2)Plate colony formation assay and soft agar colony formation assay were used to detect the colony formation ability of nasopharyngeal carcinoma 5-8F cells.(3)The tumorigenicity of nasopharyngeal carcinoma 5-8F cells was analyzed by nude mice transplantation experiment.(4)Western blotting was used to detect the expression of cancer stem cell related genes in 5-8F cells.Results:(1)Western blotting results showed that the expression of NF-kappa Bp65 in silent NPC 5-8F cells was significantly lower than that in blank control group and negative control group.(2)Cell plate cloning experiment and soft agar colony formation experiment show that compared with the blank control group and the negative control group,the clones formed by silencing NF-kappa Bp65 cells were smaller in size and less in number.(3)NPC 5-8F cells in each group were inoculated subcutaneously into the right forelimb of nude mice.After 40 days of routine feeding,the weight of transplanted tumors in the silent group of NF-kappa Bp65 was(0.047±0.094)g significantly lower than(1.793±0.628)g in the negative control group and(1.335±0.528)g in the blank control group(P < 0.05).Western blotting results showed that the expression of tumor stem cell related genes beta-catenin,CD44,Sox2 and Oct4 in the silenced group of NF-kappa Bp65 was significantly lower than that in the blank control group and the negative control group.Conclusion:(1)The protein expression of NF-?Bp65 in nasopharyngeal carcinoma 5-8F cells can be down-regulated by transfection of NF-?Bp65 siRNA plasmid expression vector.(2)The down-regulation of stem cell-related gene expression induced by silencing of NF-kappa Bp65 may be the molecular cause of inhibiting clone formation and reducing tumorigenicity of nasopharyngeal carcinoma 5-8F cells.