马立克病病毒编码的miR-M11-5p宿主靶基因的筛选与鉴定

doi:10.11843/j.issn.0366-6964.2019.05.013

畜牧兽医学报 ›› 2019, Vol. 50 ›› Issue (5): 1026-1038.doi: 10.11843/j.issn.0366-6964.2019.05.013

马立克病病毒编码的miR-M11-5p宿主靶基因的筛选与鉴定

刘豪丽^1,2, 滕蔓², 李会珍^2,3, 余祖华¹, 刘金玲², 丁轲¹, 张改平^2,3,4, 罗俊^1,2*

1. 河南科技大学动物科技学院, 动物疫病与公共卫生重点实验室, 洛阳 471003;
2. 河南省农业科学院动物免疫学重点实验室, 农业部动物免疫学重点实验室, 河南省动物免疫学重点实验室, 郑州 450002;
3. 河南农业大学牧医工程学院, 郑州 450002;
4. 扬州大学江苏高校动物重要疫病与人兽共患防控协同创新中心, 扬州 225009

收稿日期:2018-10-30 出版日期:2019-05-23 发布日期:2019-05-23
通讯作者: 罗俊,主要从事病毒分子生物学与分子致病机制研究,E-mail:luojun593@aliyun.com
作者简介:刘豪丽(1993-),女,河南安阳人,硕士,主要从事病毒分子致病机制研究,Tel:0371-65756056,E-mail:liuhaoli0708@foxmail.com
基金资助:
国家自然科学基金（31602050；U1604232）；中原千人计划-中原基础研究领军人才；河南省农业科学院杰出青年科技基金（2019JQ01）；河南省农业科学院科研发展专项资金（2019CY012）

Screening and Identification of Host mRNA Targets for the Viral microRNA miR-M11-5p Encoded by Marek's Disease Virus

LIU Haoli^1,2, TENG Man², LI Huizhen^2,3, YU Zuhua¹, LIU Jingling², DING Ke¹, ZHANG Gaiping^2,3,4, LUO Jun^1,2*

1. Key Laboratory of Animal Disease and Public Safety, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang 471003, China;
2. Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China;
3. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
4. Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonose, Yangzhou University, Yangzhou 225009, China

Received:2018-10-30 Online:2019-05-23 Published:2019-05-23

摘要/Abstract

摘要：

作为α疱疹病毒的重要成员，马立克病病毒（MDV）感染鸡可引起免疫抑制及快速发作的T细胞淋巴瘤，即马立克病（MD）。此前发现MDV基因组编码大量病毒miRNA，可能在病毒复制、潜伏感染及肿瘤发生中起重要作用。基因敲除miR-M11-5p可显著增强MDV致病性及致瘤性，表明其可能是MD肿瘤抑制因子。为进一步揭示miR-M11-5p介导的调控机制，本研究利用鸡胚成纤维细胞（CEF）总RNA的cDNA为模板，通过hybrid-PCR扩增miR-M11-5p的候选宿主靶基因片段，然后连接至pMD19-T载体、转化至E.coli JM109中构建cDNA文库。对文库菌落进行PCR鉴定、测序分析以及Blast序列对比，共获得77个候选宿主靶基因，其中37个miRNA结合靶点位于候选靶基因的3'-UTR中。进一步通过双荧光素酶报告试验、miR-M11-5p过表达以及RT-qPCR分析，最终鉴定鸡MAFB、LOC776816和RFX7为miR-M11-5p的宿主靶基因。本研究为进一步阐明miR-M11-5p在MDV肿瘤发生中的调控机制奠定了重要基础。

Abstract:

As one of the most important Alphaherpesvirus, Marek's disease virus (MDV) causes immunosuppression and rapid-onset of T-cell lymphoma in its natural host chicken, namely Marek's disease (MD). A large number of viral microRNAs (miRNAs) have been identified in the MDV genomes, which may play critical roles in virus replication, latency, and tumourigenesis. It has been previously confirmed that deletion of miR-M11-5p from the viral genome significantly enhances MDV pathogenicity and oncogenicity, suggesting that it may be a tumor suppressor during MDV tumourigenesis. To further reveal the regulatory mechanism mediated by miR-M11-5p in MDV oncogenesis, we have presently performed a hybrid-PCR to amplify the candidate host target genes using the cDNA derived from chicken embryo fibroblasts (CEF) cellular RNA as template. The PCR products were ligated into the pMD19-T vector and transformed into E. coli JM109 to construct a cDNA library for screening the candidate targets for miR-M4-5p. Based on PCR identification, DNA sequencing and BLAST analysis, a total of 77 candidate genes were obtained, of which 37 binding sites recognized by miR-M11-5p were located in the 3'-UTRs of the mRNA genes. The further analysis of dual luciferase reporter assay (DLRA), miRNA over-expression and RT-qPCR confirmed that three host genes (MAFB, LOC776816 and RFX7) were the final target genes for miR-M11-5p. Our data provide an important foundation for further elucidation of the regulatory mechanism mediated by miR-M11-5p in MDV tumourigenesis.

中图分类号:

S852.659.1

刘豪丽, 滕蔓, 李会珍, 余祖华, 刘金玲, 丁轲, 张改平, 罗俊. 马立克病病毒编码的miR-M11-5p宿主靶基因的筛选与鉴定[J]. 畜牧兽医学报, 2019, 50(5): 1026-1038.

LIU Haoli, TENG Man, LI Huizhen, YU Zuhua, LIU Jingling, DING Ke, ZHANG Gaiping, LUO Jun. Screening and Identification of Host mRNA Targets for the Viral microRNA miR-M11-5p Encoded by Marek's Disease Virus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1026-1038.

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马立克病病毒编码的miR-M11-5p宿主靶基因的筛选与鉴定

Screening and Identification of Host mRNA Targets for the Viral microRNA miR-M11-5p Encoded by Marek's Disease Virus

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