猪miR-204组织表达与重要靶基因筛选

doi:10.3969/j.issn.1004-1524.2020.09.05

浙江农业学报 ›› 2020, Vol. 32 ›› Issue (9): 1564-1573.DOI: 10.3969/j.issn.1004-1524.2020.09.05

猪miR-204组织表达与重要靶基因筛选

王伟¹, 滚双宝^1,2,*, 王鹏飞¹, 黄晓宇¹, 谢开会¹, 雒瑞瑞¹, 高小莉¹, 张博¹, 闫尊强¹, 杨巧丽¹, 马艳萍³

1.甘肃农业大学动物科学技术学院,甘肃兰州 730070;
2.甘肃省现代养猪技术工程研究中心,甘肃兰州 730070;
3.甘肃农业大学图书馆,甘肃兰州 730070

收稿日期:2020-02-17 出版日期:2020-09-25 发布日期:2020-10-10
通讯作者: *滚双宝,E-mail:gunsb@gsau.edu.cn
作者简介:王伟(1995—),男,甘肃武威人,硕士研究生,主要从事动物遗传育种与繁殖研究。E-mail:wangw@st.gsau.edu.cn
基金资助:
国家自然科学基金(31660646);甘肃省现代农业产业技术体系猪鸡产业(GARS-ZJ-1)

Tissue expression and significant target genes analysis of swine miR-204

WANG Wei¹, GUN Shuangbao^1,2,*, WANG Pengfei¹, HUANG Xiaoyu¹, XIE Kaihui¹, LUO Ruirui¹, GAO Xiaoli¹, ZHANG Bo¹, YAN Zunqiang¹, YANG Qiaoli¹, MA Yanping³

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. Gansu Agricultural University Library, Lanzhou 730070, China

Received:2020-02-17 Online:2020-09-25 Published:2020-10-10

摘要/Abstract

摘要： 基于Illumina Hiseq 4000高通量测序技术筛选得到仔猪感染C型产气荚膜梭菌耐受和易感组中显著上调表达的miR-204。为研究miR-204在各组织中的表达,进一步筛选miR-204参与调控仔猪C型产气荚膜梭菌感染的关键靶基因,采用实时荧光定量PCR方法检测了miR-204在7日龄仔猪各组织中的表达,利用MEGA7.0软件对其成熟体序列保守性进行分析,运用TargetScan、miRDB、PicTar软件进行靶基因预测,并用DAVID软件对靶基因进行Gene Ontology和KEGG Pathway通路富集分析,进一步采用qRT-PCR方法对筛选得到的关键靶基因在猪肠上皮细胞(IPEC-J2)中进行靶向关系验证。结果表明,miR-204在7日龄仔猪肾脏、肝脏、胸腺、淋巴、十二指肠和回肠组织中均高度表达,其成熟体序列在脊椎动物间高度保守。3种软件预测到的共同靶基因有114个,这些靶基因显著(P<0.05)富集在15个GO功能和13个信号通路。结合前期得到的抗性基因,筛选到4个关键靶基因NR3C1、SIRT1、BCL2L2和DLG5,转染miR-204 mimics后,靶基因SIRT1、BCL2L2和DLG5极显著(P<0.01)下调表达。miR-204是参与调控仔猪抵抗C型产气荚膜梭菌感染的重要因子,SIRT1、BCL2L2和DLG5可能是miR-204的关键靶基因,其功能还需进一步验证。

关键词: 猪, miR-204, 组织表达, 靶基因, 产气荚膜梭菌

Abstract: Based on the earlier research, miR-204 was screened out with significantly up-regulated expression in the resistance and susceptibility group for piglets infected with Clostridium perfringens type C by using Illumina Hiseq 4000 high-throughput sequencing technology. In the present study, real-time quantitative PCR was used to detect the miR-204 expression in different tissues of piglets at 7-day-old. Meanwhile the mature gene sequences was analyzed using MEGA7.0 software, and target genes were predicted using TargetScan, miRDB, and PicTar. Then, the online software DAVID was used to perform gene ontology and KEGG pathway enrichment analysis of target genes, and qRT-PCR method was used to verify the targeted relationship of the selected target genes in porcine intestinal epithelial cells (IPEC-J2). The results showed that miR-204 was highly expressed in kidney, liver, thymus, lymph, duodenum and ileum tissues of 7-day-old piglets, and its mature gene sequences was highly conserved among vertebrates. There were 114 common target genes predicted by the three softwares, and these target genes were significantly (P<0.05) enriched in 15 GO functions and 13 signaling pathways. Combined with the previously obtained resistance genes, the four key target genes nuclear receptor subfamily 3 group C member 1(NR3C1), Silence information regulator 1 (SIRT1), BCL2 like 2(BCL2L2) and Discs, large homolog 5(DLG5) were screened. After transfection of miR-204 mimics, the target genes SIRT1, BCL2L2 and DLG5 were significantly (P<0.01)down-regulated. In summary, miR-204 is an important factor involved in regulating the piglets' resistance caused by Clostridium perfringens type C infection. The genes SIRT1, BCL2L2, and DLG5 may be key target genes of miR-204, and the further study should be conducted to testify their functions.

Key words: pig (Sus scrofa), miR-204, tissue expression, target gene, Clostridium perfringens

中图分类号:

S828

王伟, 滚双宝, 王鹏飞, 黄晓宇, 谢开会, 雒瑞瑞, 高小莉, 张博, 闫尊强, 杨巧丽, 马艳萍. 猪miR-204组织表达与重要靶基因筛选[J]. 浙江农业学报, 2020, 32(9): 1564-1573.

WANG Wei, GUN Shuangbao, WANG Pengfei, HUANG Xiaoyu, XIE Kaihui, LUO Ruirui, GAO Xiaoli, ZHANG Bo, YAN Zunqiang, YANG Qiaoli, MA Yanping. Tissue expression and significant target genes analysis of swine miR-204[J]. , 2020, 32(9): 1564-1573.

参考文献

[1] AMBROS V.The functions of animal microRNAs[J]. Nature, 2004, 431(7006):350-355.
[2] BARTEL D P.MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 2004, 116(2):281-297.
[3] XIE S S, LI X, QIAN L L, et al.An integrated analysis of mRNA and miRNA in skeletal muscle from myostatin-edited Meishan pigs[J]. Genome, 2019, 62(5):305-315.
[4] ZHANG Z, WANG W, LIU J B, et al.Ssc-miR-204 regulates porcine preadipocyte differentiation and apoptosis by targeting TGFBR1 and TGFBR2[J]. Journal of Cellular Biochemistry, 2020, 121(1):609-620.
[5] WANG Q, QI R L, WANG J, et al.Differential expression profile of miRNAs in porcine muscle and adipose tissue during development[J]. Gene, 2017, 618:49-56.
[6] WU Z C, QIN W Y, WU S, et al.Identification of microRNAs regulating Escherichia coli F18 infection in Meishan weaned piglets[J]. Biology Direct, 2016, 11(1):59.
[7] YE L, SU X M, WU Z C, et al.Analysis of differential miRNA expression in the duodenum of Escherichia coli F18-sensitive and-resistant weaned piglets[J]. PLoS One, 2012, 7(8):e43741.
[8] BAO H, KOMMADATH A, LIANG G X, et al.Genome-wide whole blood microRNAome and transcriptome analyses reveal miRNA-mRNA regulated host response to foodborne pathogen Salmonella infection in swine[J]. Scientific Reports, 2015, 5:12620.
[9] YAO M, GAO W H, TAO H X, et al.Regulation signature of miR-143 and miR-26 in porcine Salmonella infection identified by binding site enrichment analysis[J]. Molecular Genetics and Genomics, 2016, 291(2):789-799.
[10] DINH H, HONG Y H, LILLEHOJ H S.Modulation of microRNAs in two genetically disparate chicken lines showing different necrotic enteritis disease susceptibility[J]. Veterinary Immunology and Immunopathology, 2014, 159(1/2):74-82.
[11] HONG Y H, DINH H, LILLEHOJ H S, et al.Differential regulation of microRNA transcriptome in chicken lines resistant and susceptible to necrotic enteritis disease[J]. Poultry Science, 2014, 93(6):1383-1395.
[12] HUANG X Y, SUN W Y, YAN Z Q, et al.Novel insights reveal anti-microbial gene regulation of piglet intestine immune in response to Clostridium perfringens infection[J]. Scientific Reports, 2019, 9:1963.
[13] WANG P F, HUANG X Y, YAN Z Q, et al.Analyses of miRNA in the ileum of diarrheic piglets caused by Clostridium perfringens type C[J]. Microbial Pathogenesis, 2019, 136:103699.
[14] 薛无涯, 王舰梅, 夏天, 等. miR-204-5p对结直肠癌细胞增殖、侵袭、迁移及上皮间质转化的影响[J]. 西南医科大学学报, 2018, 41(2):104-110.
XUE W Y, WANG J M, XIA T, et al.Effects of miR-204-5p on proliferation, migration, invasion and epithelial-mesenchymal transition in colorectal cancer cells[J]. Journal of Southwest Medical University, 2018, 41(2):104-110.(in Chinese with English abstract)
[15] ZENG J, WEI M, SHI R, et al.MiR-204-5p/Six1 feedback loop promotes epithelial-mesenchymal transition in breast cancer[J]. Tumor Biology, 2016, 37(2):2729-2735.
[16] YUAN H F, ZHANG J H, LI F L, et al.Sinomenine exerts antitumour effect in gastric cancer cells via enhancement of miR-204 expression[J]. Basic & Clinical Pharmacology & Toxicology, 2019, 125(5):450-459.
[17] 马铁军, 任利, 王燕德. miR-204-5p靶向ZEB1调控EMT途径影响胃癌细胞侵袭转移的机制研究[J]. 现代消化及介入诊疗, 2019, 24(10):1088-1094.
MA T J, REN L, WANG Y D.Mechanism of miR-204-5p targeting ZEB1 to regulate EMT pathway affecting invasion and metastasis of gastric cancer cells[J]. Modern Digestion & Intervention, 2019, 24(10):1088-1094.(in Chinese with English abstract)
[18] 黄晓宇. LncRNA和mRNA对仔猪C型产气荚膜梭菌腹泻抗性的调控机制研究[D]. 兰州: 甘肃农业大学, 2018.
HUANG X Y.Regulation mechanism of long noncoding RNA (LncRNA) and mRNA on resisting to piglet diarrhea caused by Clostridium perfringens type C[D]. Lanzhou: Gansu Agricultural University, 2018.(in Chinese with English abstract)
[19] KELLY D, O'BRIEN J J, MCCRACKEN K J, Effect of creep feeding on the incidence, duration and severity of post-weaning diarrhoea in pigs[J]. Research in Veterinary Science, 1990, 49(2):223-228.
[20] 郭云涛, 张秀秀, 苗向阳. 牛miR-320a的靶基因预测及生物信息学分析[J]. 畜牧兽医学报, 2015, 46(11):1952-1960.
GUO Y T, ZHANG X X, MIAO X Y.Target gene prediction and bioinformatics analysis of bta-miR-320a[J]. Acta Veterinaria et Zootechnica Sinica, 2015, 46(11):1952-1960.(in Chinese with English abstract)
[21] LIANG Y, WANG Y, MA L, et al.Comparison of microRNAs in adipose and muscle tissue from seven indigenous Chinese breeds and Yorkshire pigs[J]. Animal Genetics, 2019, 50(5):439-448.
[22] CHEN C, DENG B, QIAO M, et al.Solexa sequencing identification of conserved and novel microRNAs in backfat of Large White and Chinese Meishan pigs[J]. PLoS One, 2012, 7(2):e31426.
[23] SHARBATI S, FRIEDLÄNDER M R, SHARBATI J, et al. Deciphering the porcine intestinal microRNA transcriptome[J]. BMC Genomics, 2010, 11(1):1-14.
[24] 吴正常, 殷学梅, 孙丽, 等. 猪miR-192/-215的组织表达谱及其关键靶基因分析[J]. 中国农业科学, 2015, 48(11):2251-2261.
WU Z C, YIN X M, SUN L, et al.Tissue expression profile and key target genes analysis of porcine miR-192 and miR-215[J]. Scientia Agricultura Sinica, 2015, 48(11):2251-2261.(in Chinese with English abstract)
[25] CHAN G, FARZAN A, DELAY J, et al.A retrospective study on the etiological diagnoses of diarrhea in neonatal piglets in Ontario, Canada, between 2001 and 2010[J]. Canadian Journal of Veterinary Research-Revue Canadienne De Recherche Veterinaire, 2013, 77(4):254-260.
[26] YAN Z Q, JIANG T T, WANG P F, et al.Circular RNA expression profile of spleen in a Clostridium perfringens type C-induced piglet model of necrotizing enteritis[J]. FEBS Open Bio, 2018, 8(10):1722-1732.
[27] STONE E M, SWANSON M J, ROMEO A M, et al.The SIR1 gene of Saccharomyces cerevisiae and its role as an extragenic suppressor of several mating-defective mutants[J]. Molecular and Cellular Biology, 1991, 11(4):2253-2262.
[28] MA R J, ZHANG Y, ZHANG L, et al.Sirt1 protects pig oocyte against in vitro aging[J]. Animal Science Journal=Nihon Chikusan Gakkaiho, 2015, 86(9):826-832.
[29] ZHANG C Q, FANG L H, LIU X P, et al.miR-22 inhibits synovial fibroblasts proliferation and proinflammatory cytokine production in RASF via targeting SIRT1[J]. Gene, 2020, 724:144144.
[30] LEI K, DAVIS R J.JNK phosphorylation of Bim-related members of the Bcl2 family induces Bax-dependent apoptosis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(5):2432-2437.
[31] 孔贺磊, 吴金恩, 郑亚东, 等. 外泌体源性oar-miR-10b在绵羊痘病毒感染绵羊睾丸细胞中的动态表达及靶基因的鉴定[J]. 动物医学进展, 2018, 39(12):42-48.
KONG H L, WU J E, ZHENG Y D, et al.Dynamic expression of exosome-derived oar-miR-10b after the ovine testicular cells infected with sheep pox virus and identification of its target genes[J]. Progress in Veterinary Medicine, 2018, 39(12):42-48.(in Chinese with English abstract)
[32] SHI G D, LIU Y, LIU T L, et al.Upregulated miR-29b promotes neuronal cell death by inhibiting Bcl2L2 after ischemic brain injury[J]. Experimental Brain Research, 2012, 216(2):225-230.
[33] STOLL M, CORNELIUSSEN B, COSTELLO C M, et al.Genetic variation in DLG5 is associated with inflammatory bowel disease[J]. Nature Genetics, 2004, 36(5):476-480.
[34] PEARCE A V, FISHER S A, PRESCOTT N J, et al.Investigation of association of the DLG5 gene with phenotypes of inflammatory bowel disease in the British population[J]. International Journal of Colorectal Disease, 2007, 22(4):419-424.
[35] 吴嘉韵, 吴森, 戴超辉, 等. 产肠毒素性大肠杆菌侵染猪肠上皮细胞后DLG5基因的表达变化分析[J]. 江苏农业科学, 2017, 45(9):127-129.
WU J Y, WU S, DAI C H, et al.Analysis of DLG5 gene expression changes after enterotoxigenic E. coli infected pig intestinal epithelial cells[J]. Jiangsu Agricultural Sciences, 2017, 45(9):127-129.(in Chinese with English abstract)
[36] 孙丽, 吴森, 吴嘉韵, 等. 猪miR-192对DLG5和ALCAM基因的靶向作用关系验证[J]. 农业生物技术学报, 2017, 25(9):1451-1459.
SUN L, WU S, WU J Y, et al.Validation of the targeting effects of swine (Sus scrofa) miR-192 on the DLG5 and ALCAM genes[J]. Journal of Agricultural Biotechnology, 2017, 25(9):1451-1459.(in Chinese with English abstract)

猪miR-204组织表达与重要靶基因筛选

Tissue expression and significant target genes analysis of swine miR-204

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