干扰素联合利巴韦林治疗对Huh7细胞微RNA122表达的影响  

Effects of interferon and ribavirin combination therapy on microRNA122 expression in Huh7 cells

作　　者：龚邦东[1] 谢青[1] 王琳[1] 项晓刚[1] 董志霞[1] 林兰意[1] 赵钢德[1] 王晖[1] 郭清[1] 

机构地区：[1]上海交通大学医学院附属瑞金医院感染科,200025

出　　处：《中华传染病杂志》2010年第3期129-133,共5页Chinese Journal of Infectious Diseases

基　　金：基金项目：国家自然科学基金资助项目（30872252）;国家十一五重大专项（2008ZX10002-005、2008ZX10002-007）;上海市科委基础研究重点项目（08JC1415300）

摘　　要：目的探讨IFN8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3d、单用不同剂量IFNB处理4h，以及IFNB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ISG54mRNA表达，而RBV单用和联合IFN8均不能以剂量依赖方式诱导IsG54mRNA表达。以U6为内参照，在RBV终浓度为3．125mg／L时，联合IFNG100U／mL和1000U／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〈O．05），提示存在协同效果，而在RBV终浓度为6．25mg／L和12．5mg／L时无协同效果。以细胞数为内参照，RBV单用和联合IFNB均能下调细胞微RNAl22表达，以RBV终浓度为3．125mg／L明显，联合IFNB10U／mL、100u／mL和1000U／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〈0．0S），亦提示存在协同效果。结论RBV在终浓度为3．125mg／L时，能协同IFN8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。Objective To explore the effects of interferombeta （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, IFNI3 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 interferomstimulated 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 IFN[8 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

关 键 词：微RNAS 利巴韦林 干扰素Β 药物协同作用 基因表达 癌 肝细胞 

分 类 号：R735.7[医药卫生—肿瘤]