马立克病病毒miR-M4-5p宿主靶基因cDNA文库的构建及鉴定

Construction and Identification of the cDNA Library of Host mRNA Targets Recognized by miR-M4-5p Encoded by Marek's Disease Virus

马立克病病毒(MDV)是少数感染自然宿主后可诱发淋巴瘤的疱疹病毒之一,是研究肿瘤发生及发展的理想动物模型。目前已报道MDV可编码数十种miRNA,其中MDV-1编码的miR-M4-5p被鉴定为宿主细胞miR-155的同源物,由于miR-155与人类多种肿瘤性疾病密切相关,这意味着miR-M4-5p可能在MDV的致瘤过程中扮演重要角色。本研究旨在构建miR-M4-5p的宿主靶基因文库并进行初步鉴定。提取鸡胚成纤维细胞(CEF)总RNA,反转录合成cDNA,用hybrid-PCR扩增候选靶基因片段连接到pMD19-T载体,转化大肠杆菌JM109感受态细胞后挑选单克隆进行PCR鉴定及测序,通过BLAST比对分析,共获得88个宿主候选靶基因序列。优先选择miRNA结合靶点位于mRNA 3′-UTR且与miR-M4-5p种子序列(2~7nt)完全互补配对的29个候选靶基因,构建报告基因载体进行双荧光素酶报告试验,通过三轮双荧光素酶报告试验最终初步鉴定5个宿主靶基因:PRICKLE1、COLA、BCAT1、ANTXR1和TECPR1,为进一步揭示miR-M4-5p的分子调控机制奠定了重要基础。

Marek＇s disease virus（MDV）is one of the oncogenic herpes viruses that can induce tumors in their natural hosts,which is considered to be an excellent model for investigating the biology,genetics,and immunology of tumorigenesis.Recently,a lot of microRNAs（miRNAs）have been identified in MDV genomes and the miR-M4-5 p encoded by MDV-1 has been identified as a viral analog of cellular miR-155.Since miR-155 is a host miRNA associated with severalcancers,miR-M4-5 p may play a critical role in MDV oncogenesis.The present work was performed to construct a cDNA library and to primarily screen the putative host mRNA targets for miR-M4-5 p.All hybrid-PCR products amplified from CEF RNA were harvested and cloned into pMD19-T vectors,then transformed into E.coli JM109 to produce a pool.The clones were selected and sequenced,using the online basic local alignment search tool（BLAST）to analyze the target sequences.A total of 88 candidate mRNA genes were obtained,29 of which contained the predicted binding miRNA sites in the 3′-UTRs,complementary to the seed sequence of miR-M4-5 p.After three rounds of dual fluorescence reporter assays（DLRA）,five host genes including PRICKLE1,COLA,BCAT1,ANTXR1 and TECPR1 were primarily identified as the biological targets for miR-M4-5 p.Our work provides an important basis for further studies on the molecular regulatory mechanism mediated by miR-M4-5 p.