马立克氏病病毒Meq基因簇miRNA的宿主靶基因筛选鉴定及其分子调控机制

MicroRNAs (miRNAs) are recently discovered small non-coding RNAs with important post-transcriptional regulatory roles in various cellular processes, including development, differentiation, and all aspects of cancer biology. Recently a large number of viral miRNAs have been reported to be encoded in the genomes of all three serotypes of Marek's disease virus (MDV) and several recent studies suggest that some of the MDV-1-encoded miRNAs have possible functions in the replication, latency, and oncogenesis of MDV. All of the MDV-1-encoded miRNAs are focused in three gene clusters, one of which is named as Meq-cluster for its location upstream of the oncogene Meq. Previously, we have constructed a serial of Meq-clustered miRNA-knocked out MDV strains using the bacterial artificial chromosomes (BAC) and Rec E/T homologous recombinant techniques and the animal experiments had showed that some of them are play important roles in regulating the phenotype of MDV pathogenesis and oncogenesis. For further revealing the biological regulatory roles of Meq-clustered miRNAs involved in the molecular pathogenesis and oncogenesis of MDV, we will focus on the prediction, screening, and characterization of their host (chicken) target mRNAs and related signal pathways. MDV-1 is one of the oncogenic herpesviruses and provided the first demonstration of the efficacy of anti-viral vaccination in the control of cancer in any species. As a consequence studies on MDV-encoded miRNAs provide an ideal model for investigating the possible regulatory roles of virus-encoded miRNAs in the biology, genetics, and immunology of tumorigenesis.

miRNA在多种生物学过程中发挥重要的基因调控功能，最新发现MDV编码数十种miRNA，它们可能参与调控病毒的复制、潜伏感染、肿瘤发生等。MDV-1编码3个miRNA基因簇，其中Meq基因簇位于最重要的原癌基因Meq上游，它们在致病及致瘤中可能发挥关键作用。此前，我们已利用细菌人工染色体技术（BAC）和Rec E/T同源重组技术成功构建了这些miRNA单基因或基因簇的缺失毒株，经动物实验发现部分miRNA调控MDV-1的致病和致瘤表型。在此基础上，本项目拟重点开展Meq-clustered miRNA的宿主（鸡）靶基因的预测、筛选、鉴定，并对其调控的信号通路进行研究，以期为进一步阐明miRNA调控MDV-1致病及致瘤的分子机制奠定基础。MDV是目前唯一可用疫苗预防的致瘤性病毒，深入研究其编码的miRNA功能亦将为阐明miRNA调控肿瘤发生和发展的生物学、遗传学、免疫学等提供极好的动物模型。

miRNA在多种生物学过程中发挥重要的基因调控功能，最新发现MDV编码数十种miRNA，可能参与调控MDV的病毒复制、潜伏感染、肿瘤发生等。MDV-1编码3个miRNA基因簇，其中Meq基因簇位于最重要的原癌基因Meq上游，此前本项目组已利用细菌人工染色体技术（BAC）和Rec E/T同源重组技术成功构建了Meq基因簇中miRNA单基因或该基因簇的缺失毒株，经动物实验发现部分miRNA调控MDV-1的致病及致瘤表型。为了进一步阐明这些miRNA调控MDV-1致病及致瘤的潜在分子机制，本项目在实施期间对MDV-1编码的Meq基因簇病毒miRNAs的宿主和病毒靶基因进行了预测、筛选和鉴定，并对其调控的信号通路进行了研究，最终鉴定了该基因簇中编码的miR-M4-5p/3p、miR-M7-5p/3p、miR-M12-5p/3p等miRNA可能调控的一批候选靶基因，并通过双荧光素酶报告实验（DLRA）、实时荧光定量PCR（qRT-PCR）、Western blot、PCR array等系列实验等对miR-M4-5p的调控机制进行了深入研究。最终研究发现在MDV-1感染细胞及宿主的过程中，miR-M4-5p靶向调控宿主LTBP1基因、显著下调LTBP1的蛋白表达水平、导致TGF-β1的分泌抑制，从而抑制TGF-β信号通路的传递，进一步激活原癌基因c-myc的过量表达，最终促进细胞增殖，这是miR-M4-5p调控MDV肿瘤发生的重要分子机制。MDV是一种可用疫苗成功预防肿瘤发生的疱疹病毒，本研究可为阐明病毒iRNA调控肿瘤发生和发展的生物学、遗传学、免疫学等提供极好的动物模型。