乙型肝炎病毒基因组转染HepG2细胞的microRNA表达研究

乙型肝炎病毒基因组转染H epG2细胞的microRN A表达研究

刘妍　张亮　戴久增　王琳　赵建晴　徐东平

【摘要】　目的　检测乙型肝炎病毒(HBV)全基因组转染对人肝母细胞瘤细胞系Hep G2细胞中microRNA(miRNA)表达的影响,为进一步探讨肝细胞中HBV复制的分子生物学机制提供平台。方法　分别提取Hep G212115细胞(转染HBV全基因组的Hep G2细胞,实验组)和其亲本Hep G2细胞(对照组)的总RNA并分离miRNA,采用含509条探针的哺乳动物miRNA表达谱芯片对两组之间差异表达的miRNA进行分析。用SAM软件针对差异miRNA筛选的标准为在两组之间荧光强度差异4倍以上,差异miRNA的整体假阳性率为0。选择其中2个miRNA,应用实时荧光定量RT2PCR方法对芯片结果进行验证。结果　Hep G212115细胞与Hep G2细胞间差异表达的miRNA共27个(占513%),其中7个表达上调,20个表达下调。miR2181d和miR215a的实时荧光定量RT2PCR验证结果与芯片表达结果基本一致。结论　肝细胞中存在着与HBV复制相关的miRNA,这些上调和下调表达的miRNAs 可能参与了HBV在肝细胞中的复制。

【关键词】　肝炎病毒,乙型;微RNAs;基因表达

【中国图书资料分类号】　R512163

T he microRN A gene exp ression profiling in H epG2cells transfected w ith fu ll2long hep atitis B virus genom e Liu Y an,Zhang Liang,Dai Jiuzeng,et al1Viral Hepatitis Research Center,Institute of Infectious Diseases,302 Hospital of PLA,Beijing100039,China

【Abstract】　Objective　MicroRNA(miRNA)has recently been suggested to play a role in certain virus infections.The present study is to investigate if miRNA is associated with hepatitis B virus(HBV)activity by detecting differential expression of miRNA between human hepatoblastoma cell line Hep G2.2.15transfected with full2long HBV genome and its parent cell line Hep G2.M ethods　T otal RNA were extracted from Hep G2.2.15cells and control Hep G2cells,respectively.miRNAs were then isolated from the total RNA.Mammalian miRNA microarrays containing509miRNA genes were employed to analyze the differentially expressed miRNAs between the two groups. miRNAs were considered to be up2or down2regulated when the fluorescent intensity ratio between the two groups was over42fold and global false positive is zero using SAM program.Validation of microarray results was carried out by real2time quantitative RT2PCR(qRT2 PCR).R esu lts　C ompared with those of control Hep G2cells,a total of27miRNAs were differentially expressed(5.3%of all probes),a2 m ong which7were up2regulated and20were down2regulated in Hep G2.2.15cells.qRT2PCR verified that miR2181d expression was162 fold up2regulated and miR215a was92fold down2regulated in Hep G2.2.15cells,which was in agreement with the results of the microarray analysis.C onclusion　The findings suggest that there are HBV replication2associated miRNAs in HBV genome2transfected Hep G2.2.15 cells.These up2and down2regulated miRNAs may be involved in the life cycle of HBV replication.The knowledge is helpful for further study to discover new m olecular targets for anti2HBV therapy.

【K ey w ords】　hepatitis B virus;microRNAs;gene expression

核苷类似物和重组α2干扰素是目前临床上重要的抗乙型肝炎病毒(HBV)药物,但其疗效有限[1]。Mi2 croRNA(miRNA)是一类长约22个核苷酸的内源性非编码单链RNA分子,它广泛参与转录后的基因表达调控。在HCV和HIV等慢性病毒感染中,miRNA的重要作用已得到证实[2-4],但其在HBV感染中的作用尚未见报道。本研究采用哺乳动物miRNA表达谱芯片技术检测了转染HBV全基因组的人肝母细胞瘤细胞系Hep G2细胞中miRNA的表达,以探讨肝细胞中是否存在与HBV复制相关的miRNA,为进一步研究HBV在肝细胞中复制的分子生物学机制奠定基础。

1　材料与方法

111　主要实验材料　肝母细胞瘤细胞系Hep G2细胞、Hep G212115细胞(转染HBV全基因组的Hep G2细胞)由解放军第302医院传染病研究所病毒性肝炎研

究室细胞库保存。DMEM高糖细胞培养液、胎牛血清FBS购自G ibco公司,G418购自Merck公司,总RNA 提取试剂T rizol购自Invitrogen公司,其他生化试剂购自Sigma公司。哺乳动物miRNA表达谱芯片含有509条探针,其中包括的人、大鼠、小鼠的成熟miRNA数分别为435、196和261个(数据来自2005年10月Sanger 数据库,因人、大鼠和小鼠的miRNA具有共同序列,故取三者的并集),由生物芯片北京国家工程研究中心提供。芯片实验所用共聚焦扫描仪LuxScan10K/

【作者简介】　刘妍,医学硕士,助理研究员。主要从事病毒性肝炎分子生物学发病机制研究。已发表论文40余篇,获北京市科技进步二等奖1项,军队科技进步三等奖1项。E2mail:liuyan5360@1631com

【作者单位】　100039　北京　解放军第302医院传染病研究所病毒性肝炎研究中心(刘妍、戴久增、王琳、徐东平);生物芯片北京国家工程研究中心(张亮、赵建晴)

【通讯作者】　徐东平,E2mail:xudongpping@yahoo1com