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农业生物技术学报  2019, Vol. 27 Issue (5): 885-896    DOI: 10.3969/j.issn.1674-7968.2019.05.012
  研究论文与报告 本期目录 | 过刊浏览 | 高级检索 |
猪miR-339-3p靶基因预测分析及部分靶基因验证
王伟1, 黄晓宇1, 闫尊强1, 马晓文3, 王鹏飞1, 谢开会1, 雒瑞瑞1, 高小莉1, 马艳萍4, 滚双宝1, 2, *
1 甘肃农业大学 动物科学技术学院,兰州 730070;
2 甘肃省现代养猪工程技术研究中心,兰州 730070;
3 兰州大学 草地农业科技学院,兰州 730020;
4 甘肃农业大学 图书馆,兰州 730070
Target Gene Prediction Analysis and Partial Target Gene Verification of Pig (Sus scrofa) miR-339-3p
WANG Wei1, HUANG Xiao-Yu1, YAN Zun-Qiang1, MA Xiao-Wen3, WANG Peng-Fei1, XIE Kai-Hui1, LUO Rui-Rui1, GAO Xiao-Li1, MA Yan-Ping4, GUN Shuang-Bao1, 2, *
1 College of Animal Science and Technology, Gansu Agricultural University, Lanzhou 730070, China;
2 Gansu Research Center for Swine Production Engineering and Technology, Lanzhou 730070, China;
3 College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, China;
4 Gansu Agricultural University Library, Lanzhou 730070, China
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摘要 miRNAs (microRNAs)能够在多种生理和病理过程中发挥作用,通过相应的靶基因发挥其生物学功能,通过生物信息学预测和实验验证相结合的方法,可以快速、有效地筛选miRNA靶基因。本课题组前期通过高通量测序发现,ssc-miR-339-3p在仔猪(Sus scrofa)感染C型产气荚膜梭菌(Clostridium perfringens type C)对照组和攻毒组之间发生差异表达,推测其可能在感染过程中发挥了重要作用。本研究旨在对ssc-miR-339-3p的靶基因进行生物信息学预测和分析,并对ssc-miR-339-3p和预测所得部分靶基因进行验证,探索其影响和调控仔猪腹泻的可能作用机制。利用miRBase、Ensemble、NCBI、miRTarBase等数据库和PITA、RNAhybrid、miRanda、Promoter Scan、Alibaba2.1、DAVID、Cytoscape等生物信息学软件对ssc-miR-339-3p进行转录因子结合位点分析预测、不同物种间保守性分析、靶基因预测、基因本体论(Gene Ontology, GO)富集分析和京都基因与基因组百科全书(Kyoto Encyclopedia of Genes and Genomes, KEGG)信号通路富集分析;运用qRT-PCR方法对ssc-miR-339-3p和部分靶基因mRNA表达量进行验证。结果表明,miR-339-3p在各物种间高度保守;ssc-miR-339-3p启动子区域有Sp1、AP-1、C/EBPα、NF-κB、SRF、USF等多个转录因子结合位点;获得的160个靶基因显著富集于正调控MAP激酶活性、正调控细胞凋亡、负调控RNA聚合酶启动子转录、细胞凋亡信号通路等多个生物学过程及NF-κB、PI3K-Akt、FoxO、Toll样受体等信号转导通路。qRT-PCR结果显示,ssc-miR-339-3p表达量在耐受组(resistance groups, IR)和易感组(sensitive groups, IS)显著低于对照组(control groups, IC)(P<0.05),此结果与高通量测序结果基本一致;靶基因NFKB1CD40的表达量在IR和IS组极显著高于IC组(P<0.01);TRAF3IRAK1的表达量在三组之间差异不显著(P>0.05)。由此推测,ssc-miR-339-3p受到Sp1、NF-κB、USF等多种转录因子的调控,其可能通过激活NF-κB、PI3K-Akt、FoxO、Toll样受体信号通路中的靶基因NFKB1TRAF3IRAK1CD40等参与仔猪腹泻的调控。本研究预测和初步验证所得靶基因,可为今后miR-339-3p在仔猪抵抗C型产气荚膜梭菌感染过程中的功能和调控机制提供一定的实验基础和理论依据,对进一步筛选仔猪抵抗C型产气荚膜梭菌病有效的分子遗传标记提供科学依据。
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王伟
黄晓宇
闫尊强
马晓文
王鹏飞
谢开会
雒瑞瑞
高小莉
马艳萍
滚双宝
关键词 ssc-miR-339-3p靶基因生物信息学    
Abstract:miRNAs (microRNAs) play a role in a variety of physiological and pathological processes.miRNAs play biological functions through the corresponding target genes.By combining bioinformatics prediction and experimental verification, miRNA target genes can be rapidly and efficiently screened.In our early study, high-throughput sequencing revealed that ssc-miR-339-3p was differentially expressed between control group and treatment group on piglets (Sus scrofa) infected with Clostridium perfringens type C, suggesting that it might have played an important role in the infection process.This study aimed to bioinformatics prediction and analysis of the target genes of ssc-miR-339-3p, and to verify the ssc-miR-339-3p and partial target genes, to explore its possibility influence and regulating mechanism of piglets diarrhea.In this study, bioinformatics databases such as miRBase, Ensemble, NCBI, miRTarBase, and bioinformatics software such as PITA, RNAhybrid, miRanda, Promoter Scan, Alibaba2.1, DAVID, Cytoscape, were used to perform the prediction of transcription factor binding site and conservative analysis among different species of its target genes, involved Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis.qRT-PCR was used to verify the expressions of ssc-miR-339-3p and some target genes.The results showed that miR-339-3p was highly conserved among species, and multiple transcription factor binding sites such as Sp1, AP-1, C/EBPα, NF- κB, SRF and USF, were found in the ssc-miR-339-3p promoter region.The obtained 160 target genes are significantly enriched in positive regulation of MAP kinase activity, positive regulation of apoptosis, negative regulation of RNA polymerase promoter transcription, apoptosis signaling pathway and other biological processes and participated in NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways.qRT-PCR results showed that the expression level of ssc-miR-339-3p was significantly lower in the resistance groups (IR) and sensitive groups (IS) than in the control groups (IC) (P<0.05), this result was consistent with the high-throughput sequencing results; the expression levels of the target genes nuclear factor kappa-B 1 (NFKB1) and B-cell surface antigen CD40 (CD40) were significantly higher between IR and IS groups than the IC group (P<0.01); the expression of TNF receptor-associated factor 3 (TRAF3) and interleukin 1 receptor associated kinase 1 (IRAK1) were not significantly different among the 3 groups (P>0.05).Therefore, we assumed that ssc-miR-339-3p is regulated by various transcription factors such as Sp1, NF-κB and USF, which may activate NF-κB, PI3K-Akt, FoxO and Toll-like receptors signal pathways to regulate the piglets diarrhea through the target genes NFKB1, TRAF3, IRAK1, CD40.This study predicts and preliminarily validates the obtained target genes, which may provide experimental and theoretical basis for the function and regulating mechanism of miR-339-3p in piglet's resistance to Clostridium perfringens infection, thereby it can provide a theoretical basis for searching for the effective molecular genetic markers of resistance to Clostridium perfringens type C in piglets.
Key wordsPig (Sus scrofa)    ssc-miR-339-3p    Target gene    Bioinformatics
收稿日期: 2018-10-19     
ZTFLH:  S828.2  
基金资助:国家自然科学基金(No.31660646)
通讯作者: gunsb@gsau.edu.cn   
引用本文:   
王伟, 黄晓宇, 闫尊强, 马晓文, 王鹏飞, 谢开会, 雒瑞瑞, 高小莉, 马艳萍, 滚双宝. 猪miR-339-3p靶基因预测分析及部分靶基因验证[J]. 农业生物技术学报, 2019, 27(5): 885-896.
WANG Wei, HUANG Xiao-Yu, YAN Zun-Qiang, MA Xiao-Wen, WANG Peng-Fei, XIE Kai-Hui, LUO Rui-Rui, GAO Xiao-Li, MA Yan-Ping, GUN Shuang-Bao. Target Gene Prediction Analysis and Partial Target Gene Verification of Pig (Sus scrofa) miR-339-3p. 农业生物技术学报, 2019, 27(5): 885-896.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.05.012     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I5/885
 
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