NDR1对肝癌患者预后和肝癌细胞增殖、迁移的影响

Influence of NDR1 on prognosis of patients with hepatocellular carcinoma and effect on proliferation and migration of hepatocellular carcinoma cells

目的 分析核Dbf2相关激酶1(NDR1)在肝癌中的表达和临床预后的意义,探究NDR1在肝癌细胞中的生物学功能及其调控机制。方法 利用ENCORI数据库分析NDR1在肝癌组织及正常组织中的表达水平及其与肝癌患者生存率的关系。构建MYC-NDR1真核表达载体,转染肝癌HepG2细胞,通过蛋白质印迹法(Western blot)验证其表达;利用CCK-8法、细胞克隆形成实验和划痕实验分别检测细胞增殖和迁移能力。利用ENCORI数据库分析NDR1与c-Myc表达的相关性,并利用蛋白质合成抑制剂环己酰亚胺(CHX)加药实验研究NDR1与c-Myc蛋白的关系。结果 ENCORI数据库分析显示,NDR1在肝癌组织中的表达高于正常组织且NDR1高表达患者总生存率低于低表达患者,差异有统计学意义(P<0.001);Western blot检测到MYC-NDR1蛋白条带;CCK-8法结果显示,MYC-NDR1组细胞生长速度快于空载体组(P<0.001);细胞克隆形成实验显示,MYC-NDR1组的克隆数高于空载体组(P<0.001);细胞划痕实验显示,MYC-NDR1组平均迁移距离大于空载体组(P<0.001);ENCORI数据库分析显示,NDR1与c-Myc表达有着相关性(R=0.184,P<0.001);CHX加药实验显示,在相同时间内,MYC-NDR1组中c-Myc蛋白的减少量低于空载体组。结论 NDR1在肝癌组织中高表达,与肝癌患者预后不良紧密相关,并与c-Myc基因的表达呈正相关;该研究成功构建了MYC-NDR1真核表达载体,表达产物MYC-NDR1能够增加c-Myc蛋白的稳定性,促进人肝癌细胞增殖和迁移。

Objective To analyze the expression and clinical prognostic significance of nuclear Dbf2-related kinase 1(NDR1)in hepatocellular carcinoma,and to investigate the biological function and regulatory mechanism of NDR1 in hepatocellular carcinoma cells.Methods ENCORI database was used to analyze the correlation of NDR1 and c-Myc.Cycloheximide(CHX)experiment analyzed the relationship between NDR1 and c-Myc protein stability.The expression levels of NDR1 in liver cancer tissues and normal tissues and its relationship with the survival rate of liver cancer patients were analyzed using the ENCORI database.MYC-NDR1 eukaryotic expression vector was constructed,transfected with hepatocellular carcinoma HepG2 cells,and its expression was verified by protein immuno blotting(Western blot);cell proliferation and migration ability were detected by CCK-8 assay,cell clone formation assay and scratch assay,respectively.The correlation between NDR1 and c-Myc expression was analyzed using the ENCORI database,and the relationship between NDR1 and c-Myc protein was investigated using a protein synthesis inhibitor CHX dosing assay.Results The results of the ENCORI database showed that the expression of NDR1 in liver cancer tissues was higher than that in normal tissues and the overall survival rate of patients with high NDR1 expression was lower than that of patients with low NDR1 expression,and the difference was statistically significant(P<0.001).The results of the CCK-8 assay showed that the MYC-NDR1 group grew faster than the empty vector group(P<0.001).The clone formation assay showed that the number of clones in the MYC-NDR1 group was higher than that in the empty vector group(P<0.001).The cell scratch assay showed that the mean migration distance in the MYC-NDR1 group was greater than that in the empty vector group(P<0.001).ENCORI database analysis showed that NDR1 correlated with c-Myc expression(R=0.184,P<0.001);CHX dosing assay showed that the reduction of c-Myc protein in the MYC-NDR1 group was lower than that in the empty vector group during the same time.Conclusion NDR1 is highly expressed in hepatocellular carcinoma tissues,closely correlated with poor prognosis of hepatocellular carcinoma patients,and positively correlated with the expression of c-Myc gene.The study successfully constructes MYC-NDR1 eukaryotic expression vector,and the expression product MYC-NDR1 can increase the stability of c-Myc protein and promote the proliferation and migration of human hepatocellular carcinoma cells.