CAR的表达调节对肝癌基因治疗中腺病毒载体感染效率的影响

Influence of coxsackie-adenovirus receptor expression on infection efficiency of adenovirus vector in gene therapy of hepatocellular carcinoma

目的:通过抑制Raf-MEK-ERK信号转导途径调节肝癌细胞表面柯萨奇病毒和腺病毒受体(coxsackie-adenovirus receptor,CAR)的表达,探索肝癌基因治疗中CAR表达与腺病毒(adenovirus,Ad)感染效率之间的关系。方法:选择两株人肝癌细胞株SMMC-7721及HepG2,以Raf-MEK-ERK信号转导抑制剂U0126作用后,应用Western blotting检测细胞表面CAR蛋白的表达水平。选用能表达GFP的非复制型E_1A缺陷的Ad感染人肝癌细胞SMMC-7721及HepG_2,应用FACS分析U0126处理前后Ad感染效率的变化。结果:细胞经MEK抑制剂U0126处理后,细胞膜表面CAR的表达呈明显上调趋势。FACS检测显示,经U0126处理后,在相同感染复数(10 MOI)Ad-GFP的感染下,GFP^+细胞率明显升高,SMMC-7721细胞由(71.65±6.21)%上升至(86.54±5.70)%;HepG_2细胞由(77.53±4.62)%上升至(87.06±2.83)%(均P<0.05)。结论:抑制剂U0126抑制Raf-MEK-ERK信号转导途径后,可上调肝癌细胞膜表面CAR的表达,从而导致Ad在肝癌细胞感染效率的提高。

Objective： To explore the relationship between coxsackie-adenovirus receptor （CAR） expression and the infection efficiency of adenovirus vector in gene therapy for hepatocellular carcinoma through regulating CAR expression on cells surface via inhibition of the Raf/MEK/ERK pathway. Methods： Western blotting analysis was used to examine CAR expression in hepatocellular carcinoma cells （SMMC-7721 and HepG2 ） before and after treatment with U0126, inhibitor of Raf/MEK/ERK signal transduction. SMMC-7721 and HepG2 were infected by a non-replicating, EIA-deleted adenovirus expressing EGFP （Ad-GFP）. FACS was used to analyze the infection efficiency of Ad before and after U0126 treatment. Results： The expression of CAR on cell surface had an increasing tendency after treatment with U0126. FACS analysis showed significantly increased infectivity of cells treated with the MEK inhibitor U0126 compared with untreated cells： SMMC-7721, （71.65 ±6.21 ） % →（86.54 ± 5.70 ） %, HepG2, （77.53 ± 4.62 ） % → （ 87.06 ± 2.83 ） %, when infected with Ad-GFP at the same MOI （ 10 MOI ）. Conclusion： The inhibition of Raf/MEK/ERK pathway by U0126 may up-regulate the expression of CAR in some hepatocellular carcinoma cells, which subsequently enhances the susceptibility of adenovirus infection to target cells.