| 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 |
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| 引用本文： | 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. |
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| 作者姓名： | 苏芮 滕蔓 李会珍 郑鹿平 刘豪丽 刘金玲 朱志坚 罗俊 张改平 |
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| 作者单位： | 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 |
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| 基金项目： | 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） |
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| 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。
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| 关 键 词： | MDV miRNA 宿主靶基因 肿瘤发生 hybrid-PCR |
| 收稿时间： | 2020-01-17 |
Screening and Characterization of the Host mRNA Targets for the miR-M4-5p Encoded by Marek's Disease Virus |
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| 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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| Authors: | SU Rui TENG Man LI Huizhen ZHENG Luping LIU Haoli LIU Jinling ZHU Zhijian LUO Jun ZHANG Gaiping |
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| 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. |
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| Keywords: | Marek's disease virus miRNA host candidate target gene oncogenesis hybrid-PCR |
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