摘要
目的观察BC047440基因能否通过增强NF-κB的活化而促进HepG2细胞的增殖。方法设计针对BC047440基因的小干扰RNA(siRNA),构建pGenesil-1-BC047440-siRNA真核表达载体,用脂质体法将介导siRNA的真核绿色荧光表达载体pGenesil-1-BC047440-siRNA、pGenesil-1-HK-siRNA转染人肝癌HepG2细胞建立稳定细胞株;实验分为3组:转染siRNA表达载体组、无关质粒组和未处理的亲本HepG2细胞组。采用平板克隆形成实验分析细胞的增殖活性,通过Western blot检测细胞质p50含量,EMSA检测NF-κB的核转位,荧光素酶试验检测各组细胞的NF-κB活化情况。结果细胞增殖实验显示转染siRNA组细胞增殖能力明显降低,BC047440基因沉默后的HepG2的细胞质p50含量低于野生株HepG2,NF-κB核转位低于野生株HepG2,其荧光素酶和海肾荧光素酶活性比值只有野生株的1/4。结论BC047440基因能促进肝癌细胞的增殖,其调节机制可通过NF-κB信号途径实现,提示BC047440基因可能成为抑制人肝癌细胞生长的有效靶点。
Objective To investigate whether BC047440 gene can induce HepG2 proliferation through enhancing NF-κB activity. Methods Small interfering RNAs for BC047440 gene were designed using Ambion software and constructed into pGenesil-1. The pGenesil-1-HK-shRNA and pGenesil-1-BC047440-shRNA vector were all transfected into HepG2 cells using Lipofectamine TM2000. The cell colony formation rate was measured by plate colony formation assay. The protien of NF-κB signal pathway was determined by Western blotting. Nu- clear translocation and NF-κB activity were evaluated by electrophoretic mobility shift assay （EMSA） and luciferase reporter assay, respectively. Results When the cell colony formation rates were statistically decreased in siRNA treated groups, Western blot analysis revealed decreased expression of p50 in BC047440 gene-silencing HepG2 compared with that of wild HepG2. The levels of nuclear translocation of NF-κB in BC047440 gene-silencing HepG2 were also significantly suppressed compared with that of wild HepG2, and the ratio of luciferase/Renilla Luciferase in BC047440 gene-silencing HepG2 was only 25% of wild HepG2. Conclusion BC047440 can induce HepG2 proliferation through NF-κB signal pathway, and BC047440 might be a target of inhibiting the development of hepatocellular carcinoma.
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2008年第22期2090-2092,共3页
Journal of Third Military Medical University
基金
国家自然科学基金(30570843)~~
