乙型肝炎病毒基因组转染HepG2细胞的microRNA表达研究

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HepG2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HepG2.2.15细胞(转染HBV全基因组的HepG2细胞,实验组)和其亲本HepG2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RT-PCR方法对芯片结果进行验证.结果 HepG2.2.15细胞与HepG2细胞间差异表达的miRNA共27个(占5.3%),其中7个表达上调,20个表达下调.miR-181d和miR-15a的实时荧光定量RT-PCR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.

The microRNA gene expression profiling in HepG2 cells transfected with full-long hepatitis B virus genome

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 HepG2.2.15 transfected with full-long HBV genome and its parent cell line HepG2.Methods Total RNA were extracted from HepG2.2.15 cells and control HepG2 cells,respectively.miRNAs were then isolated from the total RNA.Mammalian miRNA microarrays containing 509 miRNA genes were employed to analyze the differentially expressed miRNAs between the two groups.miRNAs were considered to be up-or down-regulated when the fluorescent intensity ratio between the two groups was over 4-fold and global false positive is zero using SAM program.Validation of microarray results was carried out by real-time quantitative RT-PCR(qRT-PCR).Results Compared with those of control HepG2 cells,a total of 27 miRNAs were differentially expressed(5.3% of all probes),among which 7 were up-regulated and 20 were down-regulated in HepG2.2.15 cells.qRT-PCR verified that miR-181d expression was 16-fold up-regulated and miR-15a was 9-fold down-regulated in HepG2.2.15 cells,which was in agreement with the results of the microarray analysis.Conclusion The findings suggest that there are HBV replication-associated miRNAs in HBV genome-transfected HepG2.2.15 cells.These up-and down-regulated miRNAs may be involved in the life cycle of HBV replication.The knowledge is helpful for further study to discover new molecular targets for anti-HBV therapy.