猪miR-192对DLG5和ALCAM基因的靶向作用关系验证

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Abstract Abstract Post-weaning diarrhea (PWD) is a very common infectious disease in pig (Sus scrofa) production, which has caused huge losses to the pig industry. MicroRNAs(miRNAs) play an important role in physiological and pathological processes by acting on the corresponding target genes. MiRNAs mainly perform complete or incomplete pairing with 3' UTR of target mRNA to degrade target mRNA or inhibit mRNA translation. Afterwards, the transcriptional expression of the target gene is regulated to affect the biological activity of the individual. Our previous studies have screened out miR-192 which may be important miRNAs for regulating Escherichia coli F18 infection in weaned piglets by high-throughput sequencing and verification test, and these studies have preliminarily screened out two key target genes discs large homolog 5 (DLG5) and activated leukocyte cell adhesion molecule (ALCAM) which regulate E. coli F18 infection. In this study, the dual-luciferase reporter system and Western blotting analysis were performed to explore whether DLG5 and ALCAM genes were under the specific regulation of miR-192. Recombinant plasmids which included miRNA target sites were constructed and were co-transfected with PRL-TK and miRNA-192 mimics, inhibitor or NC into 293 cells (miRNA-192 mimics and negative control). Cells were collected for detection of luciferase activity and protein levels after 24 h transfection. Then mRNA expression of two target genes in IPEC-J2 cells infected by E. coli was detected. The 3'UTR regions of 2 target genes were constructed into the dual-luciferase reporter vector. Then, this study successfully obtained luciferase reporter gene recombinant plasmids. The results of luciferase activity showed that miR-192 mimics could bind to binding sites of target genes and significantly inhibit luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05), and miR-192 inhibitor could significantly enhance luciferase activity of ALCAM and DLG5 3'UTR dual-luciferase reporter recombinant vector (P＜0.05). Meanwhile, Western blotting analysis further indicated the target protein levels of miRNA-192 mimics group were significantly lower than the negative control group, which verified the preceding luciferase activity results. After being infected with three kinds of E. coli, the expression of 2 target genes significantly or extremely significantly increased. The results indicated that porcine mir-192 could inhibit the expression of DLG5 and ALCAM gene via targeting those 2 genes. MiR-192 and its targets played important regulatory function in replying the invasion of E. coli, maintaining stability of intestine and regulating immunization in weaned piglets. This study provides a theoretical and experimental evidence for participating in resistance to E. coli infection via porcine mir-192 targeting DLG5 and ALCAM gene, and lays the foundation for the further studies on the molecular mechanism of weaned piglets E. coli F18 diarrhea disease, thereby it can provide a theoretical basis for searching for the effective genetic markers of resistance to colibacillosis in pigs.

Key words： Pig miRNA-192 Activated leukocyte cell adhesion molecule (ALCAM) Discs large homolog 5 (DLG5) Diarrhea

Received: 09 January 2017 Published: 06 August 2017

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	XUN Li
	WU Sen
	WU Jia-Yun
	DIAO Cheng-Xiang
	WU Ku-Long
	FU Wen-Bin

Cite this article:

XUN Li,WU Sen,WU Jia-Yun, et al. Validation of the Targeting Effects of Swine (Sus scrofa) miR-192 on the DLG5 and ALCAM Genes[J]. , 2017, 25(9): 1451-1459.

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http://journal05.magtech.org.cn/Jwk_ny/EN/ OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2017/V25/I9/1451

戴丽荷，褚晓红，路伏增，等. 2015. 靶向猪ATGL 基因的miRNA预测及鉴定[J]. 畜牧兽医学报, 46(8): 1281-1289. (Dai L H, Chu X H, Lu F Z, et al. 2015. Prediction and validation of miRNA targeting porcine ATGL gene[J]. Acta Veterinaria et Zootechnica Sinica, 46(8): 1281-1289.)
何新颖, 唐志鹏, 张亚利. 2008. 肠上皮屏障与炎症性肠病研究进展[J]. 世界华人消化杂志, 16(29): 3316-3320. (He X Y, Tang Z P, Zhang Y L. 2008. Advance in enteric epithelial barrier and infl ammatory bowel disease[J]. World Chinese Journal of Digestology, 16(29): 3316-3320.)
母治平，邓娟，王滔，等. 2012. 猪miR-369靶基因肿瘤坏死因子-α（TNF--α）的鉴定[J]. 农业生物技术学报, 20(8): 922-927. (Mu Z P, Deng J, Wang T, et al. 2012. Identification of Porcine Tumor Necrosis Factor-α(TNF-α) as the Target Gene of the miR-369 [J]. Journal of Agricultural Biotechnology, 20(8): 922-9277.)
王琪, 王云霞, 王颖, 等. 2015. microRNA-let-7家族在约克夏猪甲状腺生长发育过程中的表达分析[J]. 农业生物技术学报, 23(3):337-343. (Wang Q, Wang Y X, Wang Y, et al. 2015. Expression Analysis of let-7 microRNAs in Yorkshire Pig's(Sus scrofa) Thyroid at Different Growth Stages[J]. Journal of Agricultural Biotechnology, 23(3):337-343.)
武天慧, 彭睿, 彭惠民, 等. 2015. miR-451对肺癌细胞的增殖抑制及其靶基因的初步鉴定[J]. 第三军医大学学报, 37(18): 1823-1829. (Wu T H, Peng R, Peng H M, et al. 2015. Inhibitory effect of microＲNA-451 on proliferation in lung cancer A549 cells and primary identification for its target genes[J]. Jouanal of Third Military Medical University, 37(18): 1823-1829.)
吴正常, 殷学梅, 孙丽, 等. 2015. 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, 48(11): 2251-2261. (Wu Z C, Yin X M, Sun L, et al. 2015. Tissue Expression Profile and Key Target Genes Analysis of Porcine miR-192 and miR-215[J]. Scientia Agricultura Sinica, 48(11): 2251-2261.)
庄建良, 陈庆, 许荣誉. 2008. 活化白细胞粘附分子与肿瘤的研究进展[J]. 中国肿瘤, 17(6): 479-482. (Zhuang J L, Chen Q, Xu R Y. 2008. Research Progress in Activated Leukocyte Cell Adhesion Molecule (ALCAM) and Tumor[J]. China Cancer, 17(6): 479-482.)
Alvarez-Garcia I, Miska E A. 2005. MicroRNA functions in animal development and human disease[J]. Development, 132(21): 4653-4662.
Ambros V. 2004. The functions of animal microRNAs[J]. Nature, 431(7006): 350-355.
Anderson P. 2008. Post-transcriptional control of cytokineproduction[J]. Nature Immunology, 9(4): 353-359.
Bai L, Liang R, Yang Y, et al. 2015. MicroRNA-21 regulates PI3K/Akt/mTOR signaling by targeting TGFβI during skeletal muscle development in pigs[J]. PLoS One, 10(5): e0119396.
Bao W B, Wu SL, Musa H H, et al. 2008. Genetic variation at the alpha (1,2) fucosyltransferase (FUT1) gene in Asian wild boar and Chinese and Western commercial pig breeds[J]. Journal of Animal Breeding Genetics, 125(6): 427-430.
Bartel D P. 2004. MicroRNAs: genomics, biogenesis, mechanism, and function[J]. Cell, 116(2): 281-297.
Buning C, Geerdts L, Fiedler T, et al. 2006. DlG5 variants in inflammatory bowel disease[J]. The American Journal of Gastroenterology, 101(40): 786-792.
Chiang Y, Song Y, Wang Z, et al. 2012. microRNA-192, -194 and -215 are frequently downregulated in colorectal cancer[J]. Experimental and Therapeutic Medicine, 3(3): 560-566.
Clay C C, Maniar-Hew K, Gerriets J E, et al. 2014. Early life ozone exposure results in dysregulated innate immune function and altered microRNA expression in airway epithelium[J]. PLoS One, 9(3): e90401.
Esau C, Kang X, Peralta E, et al. 2004. MicroRNA-143 regulates adipocyte differentiation[J]. Journal of Biological Chemistry, 279(50): 52361-52365.
Georges S A, Biery M C, Kim S Y, et al. 2008. Coordinated regulation of cell cycle transcripts by p53-inducible microRNAs, miR-192 and miR-215[J]. Cancer Research, 68(24): 101-105.
Hwang H W, Mendel J T. 2006. MicroRNAs in cell proliferation, cell death, and tumorigenesis[J]. British J ournal of Cancer, 94(6): 776—780.
Jin Z, Selaru F M, Cheng Y, et al. 2011. MicroRNA-192 and -215 are upregulated in human gastric cancer in vivo and suppresss AlCAM expression in vitro[J]. Oncogene, 30(13): 1577-1585.
Lagos-Quintana M, Rauhut R, Meyer J, et al. 2003. New microRNAs from mouse and human[J]. RNA, 9(2): 175-179.
Liu J, Li J, Ren Y, et al. 2014. DLG5 in cell polarity maintenance and cancer development[J]. International J ournal of Biologicl Sciences, 10(5): 543-549.
Liu X, Zhu L, Liao S, et al. 2015. The porcine microRNA transcriptome response to transmissible gastroenteritis virus infection[J]. PLoS One, 10(3): e0120377.
Loveday E K, Svinti V, Diederich S, et al. 2012. Temporal- and strain-specific host microRNA molecular signatures associated with swine-origin H1N1 and avian-origin H7N7 influenza A virus infection[J]. Journal of Virology, 86(11): 6109-6122.
Meijerink E, Neuenschwander S, Fries R, et al. 2000. A DNA polymorphism influencing alpha(1,2) fucosyltransferase activity of the pig FUT1 enzyme determines susceptibility of small intestinal epithelium to Eacherichia coli F18 adhesion[J]. Immunogenetics, 52(1-2): 129-136.
Nagy B, Fekete P Z. 1999. Enterotoxigenic Escherichia coli (ETEC) in farm animal[J]. Veterinary Research, 30(2-3): 259-284.
Sezaki T, Tomiyama L, Kimura Y, et al. 2013. Dlg5 interacts with the TGF-β receptor and promotes its degradation[J]. FEBS Letters, 587(11): 1624-1629.
Stoll M, Corneliussen B, Costello C M, et al. 2004. Genetic variation in DLG5 is associated with inflammatory bowel disease[J]. Nature Genetics, 36(5): 476-480.
Tan F, Mbunkui F, Ofori-Acquah S F. 2012. Cloning of the human activated leukocyte cell adhesion molecule promoter and identification of its tissue-independent transcriptional activation by Sp1[J]. Cellular & Molecular Biology Letters, 17(4): 571-585.
Vogeli P, Meijerink E, Fries R, et al. 1997. A molecular test for the detection of E. coli F18 Receptors: a breakthrough in the struggle against Oedema disease and postweaning Diarrhoea in swine[J]. Schweizer Archiv für Tierheilkunde, 139(11): 479-484.
Wang B, Herman-Edelstein M, Koh P, et al. 2010. E-cadherin expression is regulated by miR-192 /215 by a mechanism that is independent of the profibrotic effects of transforming growth factor- beta[J]. Diabetes, 59 (7 ) : 1794-1802．
Wienholds E, Plasterk R H. 2005. MicroRNA function in animal development[J]. FEBS Letters, 579(26): 5911-5922.
Xia B, Song H, Chen Y, et al. 2013. Efficient inhibition of porcine reproductive and respiratory syndrome virus replication by artificial microRNAs targeting the untranslated regions[J]. Archives Virology, 158(1): 55-61.
Ye L, Su X M, Wu Z C, et al. 2012. Analysis of differential miRNA expression in the duodenum of Escherichia coli F18-sensitive and -resistant weaned piglets[J]. PLoS One, 7(8): e43741.