马立克病病毒编码的miR-M4-5p调控宿主靶基因的筛选及鉴定

doi:10.11843/j.issn.0366-6964.2020.08.020

畜牧兽医学报 ›› 2020, Vol. 51 ›› Issue (8): 1956-1969.doi: 10.11843/j.issn.0366-6964.2020.08.020

马立克病病毒编码的miR-M4-5p调控宿主靶基因的筛选及鉴定

苏芮^1,2,3, 滕蔓^2,3, 李会珍^1,2,3, 郑鹿平^2,3, 刘豪丽^2,3,4, 刘金玲^2,3, 朱志坚^2,3, 罗俊^2,3,4, 张改平^1,2,5*

1. 河南农业大学牧医工程学院, 郑州 450002;
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. 河南省农业科学院, 中英禽病国际研究中心, 郑州 450002;
4. 河南科技大学动物科技学院, 动物疫病与公共安全重点实验室, 洛阳 471003;
5. 扬州大学江苏高校动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2020-01-17 出版日期:2020-08-25 发布日期:2020-08-19
通讯作者: 张改平,主要从事动物免疫学及重大疫病快速检测技术研究,E-mail:zhanggaip@126.com
作者简介:苏芮(1995-),女,河南卫辉人,硕士生,主要从事病毒分子致病机制研究,Tel:0371-65756056,E-mail:919608211@qq.com
基金资助:
国家自然科学基金（U1604232；31602050）；中原千人计划-中原基础研究领军人才；河南省重点研发与推广专项（192102110072）；河南省农业科学院杰出青年科技基金（2019JQ01）

Screening and Characterization of the Host mRNA Targets for the miR-M4-5p Encoded by Marek's Disease Virus

SU Rui^1,2,3, TENG Man^2,3, LI Huizhen^1,2,3, ZHENG Luping^2,3, LIU Haoli^2,3,4, LIU Jinling^2,3, ZHU Zhijian^2,3, LUO Jun^2,3,4, ZHANG Gaiping^1,2,5*

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

Received:2020-01-17 Online:2020-08-25 Published:2020-08-19

摘要/Abstract

摘要： 血清1型马立克病病毒（MDV-1）感染引起的鸡马立克病（MD）一直以来被认为是研究病毒诱导肿瘤发生机制的理想模型。此前研究发现，MDV-1编码的miR-M4-5p是宿主癌基因miR-155的病毒同源物，从基因组中敲除miR-M4-5p可显著降低MDV-1的致病性和致瘤性，表明miR-M4-5p可能是MDV-1诱导肿瘤发生的重要调控因子。为进一步揭示miR-M4-5p的调控机制，以CEF细胞总RNA反转录产物cDNA为模板，利用hybrid-PCR技术构建了miR-M4-5p的候选靶基因文库。通过基因克隆、PCR鉴定及序列比对分析，获得128个候选基因序列，有73个基因的3'-UTR存在miR-M4-5p的潜在结合靶点，其中23个3'-UTR结合靶点与miR-M4-5p完全互补配对。通过双荧光素酶报告试验和qRT-PCR分析，对miR-M4-5p与3'-UTR的体内外相互作用以及候选靶基因的表达水平进行分析和验证，最终鉴定DPT、TMEM230和DCLK1为miR-M4-5p调控的宿主靶基因。本研究为后续进一步阐明miR-M4-5p在MD肿瘤发生中的调控机制奠定了重要基础。

关键词: MDV, miRNA, 宿主靶基因, 肿瘤发生, hybrid-PCR

Abstract: Marek's disease, a rapid-onset neoplastic disease in poultry caused by the serotype 1 Marek's disease virus (MDV-1), has been historically regarded as an ideal model for the research on virally-induced cancers. Previous studies have demonstrated that the deletion of the miR-M4-5p, a viral analog of cellular oncogene miR-155 encoded by MDV-1, from the viral genome significantly reduces virus pathogenicity and oncogenicity, suggesting an important regulatory role responsible for MD tumorigenesis. To further reveal the regulatory mechanism mediated by miR-M4-5p in MD biology, we used CEF total cellular RNA as the template to produce a cDNA library utilizing the hybrid-PCR, for screening the host mRNA targets for miR-M4-5p. A total of 128 candidate host genes were obtained, of which there are 73 candidate binding sites were located in the 3'-UTRs and 23 of them contain the perfect matches of miRNA:mRNA. Further experiments of the dual luciferase reporter assay (DLRA) and the quantitative real-time PCR (qRT-PCR) analysis characterized three host genes, DPT, TMEM230 and DCLK1, as the in vivo targets for miR-M4-5p. Our work provides an important basis for further elucidating the regulatory mechanism of miR-M4-5p in MD oncogenesis.

Key words: Marek's disease virus, miRNA, host candidate target gene, oncogenesis, hybrid-PCR

中图分类号:

S852.659.1

苏芮, 滕蔓, 李会珍, 郑鹿平, 刘豪丽, 刘金玲, 朱志坚, 罗俊, 张改平. 马立克病病毒编码的miR-M4-5p调控宿主靶基因的筛选及鉴定[J]. 畜牧兽医学报, 2020, 51(8): 1956-1969.

SU Rui, TENG Man, LI Huizhen, ZHENG Luping, LIU Haoli, LIU Jinling, ZHU Zhijian, LUO Jun, ZHANG Gaiping. Screening and Characterization of the Host mRNA Targets for the miR-M4-5p Encoded by Marek's Disease Virus[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(8): 1956-1969.

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马立克病病毒编码的miR-M4-5p调控宿主靶基因的筛选及鉴定

Screening and Characterization of the Host mRNA Targets for the miR-M4-5p Encoded by Marek's Disease Virus

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