miR-181a在转染乙型肝炎病毒基因组的nepG2.2.15细胞中的表达研究

目的 鉴定分析microRNA(miRNA)miR-181a在转染了乙型肝炎病毒(HBV)基因组的HepG2.2.15细胞中的表达情况,探讨miR-181a在与HBV相关的肝脏疾病中的作用.方法 以本课题组基因芯片结果为基础,设计并合成miR-181a探针,采用Northern blotting检测miR-181a在HepG2.2.15和HepG2细胞(对照组)中的表达水平,应用生物信息学方法结合mRNA表达谱芯片结果预测miR-181a的靶基因,选取靶基因HLA-A2,应用流式细胞仪分析HLA-A2分子在上述2种细胞中的表达.结果 Northernblotting结果显示,与对照组相比,miR-181a在HepG2.2.15细胞中的表达量显著增高;miR-181a可能的靶基因包括C8A、IDH1和HLA-A等,miR-181a可能通过其种子序列与其靶基因HLA-A的3'-UTR区部分互补结合来调节HLA-A的表达;流式分析也显示HLA-A2分子在HepG2.2.15细胞中的表达量(43.9%)显著低于HepG2细胞(96.6%).结论 miR-181a在HBV转染的HepG2.2.15细胞中高表达,并可能下调靶基因HLA-A的表达,推测这可能是HBV感染后病毒逃避免疫反应、持续复制的机制之一.

Up-regulated expression of miR-181a in HepG2.2.15 cells transfected via full-length hepatitis B virus genome

Objective To identify and analyze the expression of microRNA miR-181a in HepG2.2.15 cell line transfected by full-length hepatitis B virus genome,and explore the potential role of miR-181a engaged in the development of HBV-related liver disease.Methods Based on the previous data from microRNA microarray,miR-181a specific probe was designed and synthesized.The expressive levels of miR-181a in HepG2.2.15 and its parent cell line HepG2 were analyzed using Northern blot analysis.Some putative targets of miR-181a were predicted by computational software RNAhybrid and confirmed by mRNA microarray.One of the targets was selected and flow cytometry analysis was used to further determine the difference of intracellular HLA-A2 level between the two cell lines.Results Northern blot analysis showed that the expressive level of miR-181a in HepG2.2.15 cell was significantly up-regulated compared with that in HepG2 cell.Some putative targets of miR-181a including C8A,IDH1 and HLA-A were predicted and miR-181a might down-regulate the expression of HLA-A gene via partial complement to the 3'-UTR of HLA-A gene.Consistency with the result of mRNA profile microarray,FCM analysis also showed a significantly lower expression of HLA-A2(43.9%)in HepG2.2.15 cell than that in HepG2 cell(96.6%).Conclusion The expressive level of miR-181a in HepG2.2.15 cell is significantly up-regulated and miR-181a might down-regulate its target gene HLA-A,which might be one of the molecular mechanisms that HBV may escape from the immune response and continue replication in hepatocytes.The knowledge is also helpful for understanding the mechanism of HBV-host microRNA interaction.