马立克氏病病毒（MDV）编码的miRNA抑癌基因的发现与鉴定

In recent several years, a total of 26 viral miRNAs focusing in three gene clusteres namly the Meq-, Mid- and LAT-clusters have been reported in the genomes of Marek’s disease virus serogype 1 (MDV-1). Some of the MDV-1 miRNAs have been suggested to play important regulatory functions in the virus replication, latency maintenance and oncogenesis of MDV. In a previous work, we have found that completely knock out the Mid-cluster can significantly increase the motility and oncogenicity of MDV-1, implying that some of the Mid-clustered miRNAs may potentially play as tumor suppresor gene(s) in MDV oncogenesis. In the present project, in order to identify the MDV-1-encoded tumor suppressor miRNA gene(s), we plan to independantly construct a serial of all the three Mid-clustered miRNA precursor-deleted MDV strains, using the bacterial artificial chromosomes (BAC) and Rec E/T homologous recombinant techniques, and further identify the tumor suppressor miRNA gene(s) by animal challenge experiments. Furthormore, the host mRNA targets of identified tumor suppressor miRNA gene(s) will be simultaneiously predicted and characterized based on the methods of bioinformatic analysis, 3’-full RACE/5’-anchor PCR, Dual-Luciferase Reportor Assay (DLRA) and quantitative real-time PCR (qRT-PCR). Our study will provides an important basis for further eluciating the molecular regulatory mechanisms mediated by the tumor suppressor miRNA gene(s) in MD lymphomagenesis.

血清1型马立克氏病病毒（MDV-1）共编码26个miRNA，在基因组中形成Meq、Mid和LAT 3个基因簇，其中部分病毒miRNA在MDV的复制、潜伏感染及致瘤过程中可能发挥重要调控作用。申请人近期研究发现，将整个Mid基因簇miRNA进行基因缺失可显著增强MDV的致死率和致瘤性，预示该基因簇中某些miRNA可能是MD肿瘤发生的抑癌基因。本项目拟利用细菌人工染色体（BAC）和Rec E/T同源重组技术，进一步构建Mid基因簇内编码的3个miRNA前体的单一基因缺失毒株，然后通过动物实验发现鉴定抑制MDV致瘤的miRNA候选基因，最后结合生物信息学预测、3’-Full RACE/5'-锚定PCR建库测序、双荧光素酶报告实验（DLRA）、实时荧光定量PCR（qRT-PCR）等实验对抑癌miRNA的宿主靶基因进行鉴定，为后续阐明miRNA抑癌基因在MD肿瘤发生中的分子调控机制奠定基础。

血清1型马立克病病毒（MDV-1）是家禽最重要的致肿瘤性病原之一。该病毒编码26个miRNA，在基因组中形成Meq、Mid和LAT 3个基因簇。我们前期的研究发现，整个Mid基因簇miRNA的缺失可显著增强MDV-1的致死率和致瘤性，预示该基因簇中某些miRNA可能是MD肿瘤发生的抑癌基因。本项目主要研究内容是利用细菌人工染色体（BAC）和Rec E/T同源重组技术，构建Mid基因簇内编码的3个miRNA前体的单一基因缺失毒株，通过动物实验发现和鉴定抑制MDV-1致瘤的miRNA候选基因，最后结合生物信息学预测、3’-Full RACE/5'-锚定PCR建库测序、双荧光素酶报告实验（DLRA）、实时荧光定量PCR（qRT-PCR）等实验对抑癌miRNA调控的宿主靶基因进行鉴定。项目获资助实施以来，首先利用BAC和Rec E/T同源重组技术构建了3个Mid基因簇miRNA前体单基因缺失的MDV-1毒株。动物攻毒实验结果显示，与GX0101亲本毒株相比，miR-M1基因缺失增强了MDV-1的致死率，miR-M11的基因缺失增强了MDV-1的致瘤率，而miR-M31的基因缺失则同时降低了MDV-1的致死率和致瘤率，这些研究结果表明Mid基因簇中miR-M11和miR-M31编码的miRNA可能在MDV-1致瘤过程中分别发挥抑癌或致癌基因的作用。随后项目组分别构建了miR-M11-5p和miR-M31-3p的宿主候选靶基因cDNA文库，并对候选靶基因进行了筛选和鉴定，最终将鸡MAFB、LOC776816和RFX7鉴定为miR-M11-5p调控的宿主靶基因，将鸡CNDP2、GNPAT、TCF12、FIGNL1和MGST1鉴定为miR-M31-3p的宿主靶基因，为进一步揭示这这些病毒miRNA在MD肿瘤发生中的分子调控机制奠定了重要基础。