摘要目的 探讨IFNβ联合利巴韦林(RBV)治疗对肝癌细胞株Huh7的微RNA122表达的影响.方法 Huh7细胞分别经过单用不同剂量RBV处理3 d、单用不同剂量IFNβ处理4 h,以及IFNβ联合RBV处理后,采用噻唑蓝(MTT)法检测其生长曲线,RT-PCR法检测IFN诱导基因54(ISG54)的表达,然后分别以小核核糖核酸6(U6)和单位细胞数为内参照,茎环结构实时PCR检测微RNA122表达变化.数据比较采用单因素方差分析,两两比较采用q检验.结果 MTT结果显示,RBV能以剂量依赖方式抑制Huh7细胞增殖,而IFNβ仅能轻微地且不能以剂量依赖方式抑制Huh7细胞增殖.RT-PCR结果显示,IFNβ能以剂量依赖方式诱导Huh7细胞ISG54 mRNA表达,而RBV单用和联合IFNβ均不能以剂量依赖方式诱导ISG54 mRNA表达.以U6为内参照,在RBV终浓度为3.125 mg/L时,联合lFN8 100 U/mL和1000 U/mL,微RNA122相对表达量分别为0.770±0.082和0.720±0.045,与单用IFNβ组相比,差异有统计学意义(q=4.623,q=5.112;均P<0.05).提示存在协同效果,而在RBV终浓度为6.25 mg/L和12.5 mg/L时无协同效果.以细胞数为内参照,RBV单用和联合IFNβ均能下调细胞微RNA122表达,以RBV终浓度为3.125 mg/L明显,联合IFNβ 10 U/mL、100 U/mL和1000 U/mL时,微RNA122相对表达量分别为0.680±0.055、0.560±0.084和0.610±0.030,与单用IFNβ组相比,差异有统计学意义(F=4.121,P<0.05),亦提示存在协同效果.结论 RBV在终浓度为3.125 mg/L时,能协同IFNβ下调Huh7细胞微RNA122表达,这可能为临床上RBV提高IFN抗HCV疗效的一个新的分子机制.

abstractsObjective To explore the effects of interferon-beta(IFNβ) and ribavirin (RBV) combination therapy on microRNA122 expression in human hepatoma cell line Huh7. Methods Huh7 cells were treated with RBV alone of different doses for 3 days, IFNβ alone of different doses for 4 h and IFNβ and RBV combinations. The cell growth was measured by thylthiazol blue tetrazolium bromide (MTT) assay. Expression of interferon-stimulated gene (ISG)54 was detected by reverse transcription-polymerase chain reaction (RT-PCR) and microRNA122 expression was analyzed by stem-loop real time PCR using U6 and per cell as internal controls. The data comparison was done by single factor analysis of variance, and paired comparison was done by q test. Results MTT assay showed that RBV suppressed Huh7 cell growth dose-dependently, while IFNβ only slightly suppressed in a dose-independent manner. RT-PCR showed that IFNβ induced ISG54 mRNA expression in Huh7 cells dose-dependently, while RBV combined with or without IFNβ did not induce ISG54 mRNA expression dose-dependently. When using U6 as internal control, and treated with RBV of final concentration of 3. 125 mg/L combined with IFNβ of 100 U/mL and 1000 U/mL, the relative expressions of microRNA122 were 0. 770±0. 082 and 0. 720±0. 045, respectively, which were both significantly different from IFNβ monotherapy (q=4. 623 and q=5. 112, respectively; both P<0. 05) and showed synergistic inhibitory effects. There were no synergistic effects when final concentrations of RBV were 6. 25 mg/L and 12. 5 mg/L. Whereas, when using per cell as internal control, RBV combined with or without IFNβ both downregulated microRNA122 expression and the effect of RBV with final concentration of 3. 125 mg/L was the most significant. The relative expressions of microRNA122 when treated with RBV combined with IFNβ of 10 U/mL, 100 U/mL, 1000 U/mL were 0. 680 ± 0. 055, 0. 560 ± 0. 084 and 0. 610 ± 0. 030, respectively, which were significantly different from IFNβ monotherapy (F=4. 121,P<0. 05) and also showed synergistic inhibitory effects. Conclusions RBV final concentration of 3. 125 mg/L could synergistically downregulate microRNA122 expressions in Huh7 cells with IFNβ, which may provide a novel molecular mechanism for RBV augmenting IFN response in hepatitis C virus (HCV) treatment.