IKBKB基因敲除对胃癌细胞NLRC5、NF-κB表达以及生物学功能的影响

Effects of IKBKB gene knockdown on NLRC5 and NF-κB expression,as well as the biological function of gastric cancer cells

目的:探究IKBKB基因在胃癌细胞中的表达水平与NLRC5、NF-κB的作用关系,明确其对胃癌细胞生物学功能影响。方法:采用pLenti-IKBKB-sgRNA基因敲除质粒直接对人胃癌SGC-7901细胞系进行转染,应用免疫印迹试验(western blot,WB)检测单克隆细胞株中IKBKB基因敲除的效果,以及NLRC5、NF-κB的表达水平。划痕实验和Transwell迁移实验检测单克隆细胞的迁移能力,流式细胞术检测细胞凋亡和细胞周期。成瘤实验观察单克隆细胞对瘤体生长的影响。在临床胃癌组织和癌旁组织中采用免疫组化染色评估NLRC5、NF-κB、IKBKB的表达状态。明确IKBKB与NLRC5、NF-κB的相关性以及临床诊断价值。结果:敲除IKBKB基因后人胃癌SGC-7901细胞中NLRC5、NF-κB的表达水平显著降低,细胞迁移和侵袭能力减弱。细胞发生G1期阻滞,降低了SGC-79013细胞增殖能力,细胞总凋亡率显著上升。IKBKB敲除后NLRC5、NF-κB表达水平下调影响瘤体的生长发育。NLRC5、NF-κB、IKBKB在胃癌组织中表达水平高于癌旁组织,NLRC5、NF-κB与IKBKB的表达水平呈正相关。IKBKB、NLRC5、NF-κB三者联合诊断胃癌AUC为0.921(0.876~0.967)。结论:IKBKB在胃癌细胞与NLRC5、NF-κB存在相关作用关系,协同促进胃癌的发生和发展。

Objective To investigate the relationship between the expression level of the IKBKB gene in gastric cancer cells and the activitiesy of NLRC5 and NF-κB.The effect of IKBKB knockdown on the biological function of gastric cancer cells was clarified.Methods The human gastric cancer SGC-7901 cell line was transfected directly with the plenti-ikkbkb-sgrna gene knockdownout plasmid.Western blot(WB)was used to detect the effect of Ikkbkb gene knockout and the expression levels of NLRC5 and NF-κB in the monoclonal cell line.Scratch test and Transwell migration assay were used to assess the migratory capacity of monoclonal cells,while flow cytometry was employed to measure apoptosis and cell cycle progression.The effect of monoclonal cells on tumor growth was observed in a tumorigenesis experiment.Immunohistochemical staining was used to evaluate the expression status of NLRC5,NF-κB,and IKBKB in clinical gastric cancer tissues and adjacent tissues.To clarify their existence,expression correlation,and clinical diagnostic value.Results After knocking out the IKBKB gene,the expression levels of NLRC5 and NF-κB in human gastric cancer SGC-7901 cells were significantly reduced,and the cell migration and invasion abilities were weakened.G1 phase arrest decreased the proliferation ability of SGC-79013 cells,and the total apoptosis rate of SGC-79013 cells significantly increased.The expression levels of NLRC5 and NF-κB were downregulated after IKBKB knockout,which impacted the growth and development of tumors.The expression levels of NLRC5,NF-κB,and IKBKB in gastric cancer tissues were higher than those in adjacent tissues.Additionally,the expression levels of NLRC5 and NF-κB were positively correlated with IKBKB.The binary logistic regression model was constructed with IKBKB,NLRC5,and NF-κB as explanatory variables.The diagnostic performance of the joint model was quantified using ROC analysis,which showed an AUC of 0.921 with a 95%confidence interval of 0.876-0.967.Conclusion IKBKB has a correlation with NLRC5 and NF-κB in gastric cancer cells,and synergistically promotes the occurrence and development of gastric cancer.