罗伊氏乳杆菌对仔猪空肠miRNAs表达谱的影响

doi:10.11843/j.issn.0366-6964.2020.09.013

摘要/Abstract

摘要： 试验旨在研究罗伊氏乳杆菌对仔猪空肠miRNAs表达的影响。选用1日龄健康约克×荣昌仔猪6窝，随机分为两组，每组3窝（共27头）。即对照组（每天每头灌喂0.1%的无菌蛋白胨溶液），LR组（每天每头灌喂活菌总数为1.0×10¹⁰CFU的罗伊氏乳杆菌），分别在10和20日龄，每组选取6头进行屠宰，采集空肠样品。通过Solexa高通量测序技术检测空肠miRNA的表达谱，利用生物信息学技术进行靶基因预测和功能分析。结果显示：1）试验构建了8个文库，共获得187 103 840条纯净reads序列，其中22 nt长度序列占的比例最大；2）10和20日龄分别鉴定出354和352个已知的miRNAs，其中有329个miRNAs共表达；3）灌喂罗伊氏乳杆菌组仔猪空肠miRNA差异表达，10和20日龄分别获得7和9个差异显著的miRNAs，ssc-miR-218在两个日龄中都差异表达；4）对差异表达的miRNAs进行靶基因预测和KEGG通路分析，发现有10 221个靶基因富集到293条通路上，有66条通路显著富集（P<0.05），包括与肠道发育及免疫调控相关的MAPK和PI3K-Akt信号通路；5）随机挑选6个差异表达miRNAs进行荧光定量PCR验证，验证结果与测序结果基本一致。本研究表明，罗伊氏乳杆菌可能通过影响空肠miRNAs表达来调控有关肠道健康的信号通路。

关键词: 罗伊氏乳杆菌, miRNA, Solexa高通量测序, 空肠, 仔猪

Abstract: This study was conducted to investigate the effect of Lactobacillus reuteri on the expression of miRNAs in the jejunum of piglets. Six litters of healthy 1-day-old York×Rongchang piglets were selected and randomly divided into the two group，3 litters per group(n=27). The two groups were control group (daily orally adminitrated with 0.1% sterile peptone solution per piglet)，and the LR group (daily orally administrated with 1.0×10¹⁰ CFU of L. reuteri per piglet)， respectively. At 10 and 20 days of age, six piglets per group were selected for slaughter, respectively, and the jejunum samples were collected. After RNA extraction, the library was constructed and the miRNA expression profile of the jejunum was detected by Solexa high-throughput sequencing technology. Bioinformatics technology was used to conduct target genes prediction and functional analysis. The results showed that: 1) A total of 8 libraries were constructed and a total of 187 103 840 clean reads were obtained, of which the 22 nt length sequence accounted for the largest proportion; 2) Three hundred and fifty-four and 352 known miRNAs were respectively identified at 10 and 20 days of age, of which 329 miRNAs were co-expressed; 3) Differentially expressed (DE) miRNAs in the jejunum were found after L. reuteri administration, 7 and 9 DE miRNAs were obtained at 10 and 20 days of age，respectively. Notably, ssc-miR-218 was expressed differentially at two ages. 4) KEGG pathway analysis and target gene prediction on the differentially expressed miRNAs revealed that 10 221 target genes were enriched in 293 biological pathways, of which 66 were significantly enriched (P<0.05), including the MAPK signaling pathway and PI3K-Akt signaling pathway, which were known to regulate intestinal development and immune. 5) Six miRNAs were selected to validate the sequencing results by qRT-PCR, the qRT-PCR expression results corresponded well with those from the sequencing. The results indicates that L. reuteri may regulate intestinal health related pathways by influencing the expression of jejunal miRNAs.

Key words: Lactobacillus reuteri, miRNA, Solexa high-throughput sequencing, jejunum, piglet

中图分类号:

S828

孙倩, 王琪, 黄金秀, 齐仁立, 杨飞云, 王瑞生, 兰云贤. 罗伊氏乳杆菌对仔猪空肠miRNAs表达谱的影响[J]. 畜牧兽医学报, 2020, 51(9): 2156-2164.

SUN Qian, WANG Qi, HUANG Jinxiu, QI Renli, YANG Feiyun, WANG Ruisheng, LAN Yunxian. Effect of Lactobacillus reuteri on the Expression Profile of miRNAs in the Jejunum of Piglets[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(9): 2156-2164.

参考文献 38

[1]	WEGMANN U,MACKENZIE D A,ZHENG J S,et al.The pan-genome of Lactobacillus reuteri strains originating from the pig gastrointestinal tract[J].BMC Genomics,2015,16(1):1023.
[2]	LIU H,ZHANG J,ZHANG S H,et al.Oral administration of Lactobacillus fermentum I5007 favors intestinal development and alters the intestinal microbiota in formula-fed piglets[J].J Agric Food Chem,2014,62(4):860-866.
[3]	DOWARAH R,VERMA A K,AGARWAL N,et al.Effect of swine based probiotic on performance,diarrhoea scores,intestinal microbiota and gut health of grower-finisher crossbred pigs[J].Livest Sci,2017,195:74-79.
[4]	TALARICO T L,CASAS I A,CHUNG T C,et al.Production and isolation of reuterin,a growth inhibitor produced by Lactobacillus reuteri[J].Antimicrob Agents Chemother,1988,32(12):1854-1858.
[5]	HOU C L,WANG Q W,ZENG X F,et al.Complete genome sequence of Lactobacillus reuteri I5007,a probiotic strain isolated from healthy piglet[J].J Biotechnol,2014,179:63-64.
[6]	YANG F J,WANG A N,ZENG X F,et al.Lactobacillus reuteri I5007 modulates tight junction protein expression in IPEC-J2 cells with LPS stimulation and in newborn piglets under normal conditions[J].BMC Microbiol,2015,15(1):32.
[7]	LIU Y Y,FATHEREE N Y,MANGALAT N,et al.Lactobacillus reuteri strains reduce incidence and severity of experimental necrotizing enterocolitis via modulation of TLR4 and NF-κB signaling in the intestine[J].Am J Physiol Gastrointest Liver Physiol,2012,302(6):G608-G617.
[8]	PUA H H,ANSEL K M.MicroRNA regulation of allergic inflammation and asthma[J].Curr Opin Immunol,2015,36:101-108.
[9]	BARTEL D P.MicroRNAs:target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[10]	WU L Y,MA X P,SHI Y,et al.Alterations in microRNA expression profiles in inflamed and non-inflamed ascending colon mucosae of patients with active Crohn's disease[J].J Gastroenterol Hepatol,2017,32(10):1706-1715.
[11]	MCKENNA L B,SCHUG J,VOUREKAS A,et al.MicroRNAs control intestinal epithelial differentiation,architecture,and barrier function[J].Gastroenterology,2010,139(5):1654-1664.
[12]	HOEKE L,SHARBATI J,PAWAR K,et al.Intestinal Salmonella typhimurium infection leads to miR-29a induced Caveolin 2 regulation[J].PLoS One,2013,8(6):e67300.
[13]	HEYDARI Z,RAHAIE M,ALIZADEH A M,et al.Effects of Lactobacillus acidophilus and Bifidobacterium bifidum probiotics on the expression of MicroRNAs 135b,26b,18a and 155,and their involving genes in mice colon cancer[J].Probiotics Antimicrob Proteins,2019,11(4):1155-1162.
[14]	孙倩,王琪,王敬,等.miRNAs参与肠道微生物和宿主免疫互作的调控[J].动物营养学报,2020,32(1):79-84.SUN Q,WANG Q,WANG J,et al.miRNAs regulation on interaction between gut microbiota and host immunity[J].Chinese Journal of Animal Nutrition,2020,32(1):79-84.(in Chinese)
[15]	SHARBATI S,FRIEDLANDER M R,SHARBATI J,et al.Deciphering the porcine intestinal microRNA transcriptome[J].BMC Genomics,2010,11(1):275.
[16]	TAO X,XU Z W,MEN X M.Analysis of serum microRNA expression profiles and comparison with small intestinal microRNA expression profiles in weaned piglets[J].PLoS One,2016,11(9):e0162776.
[17]	ARDILA H J,SANABRIA-SALAS M C,MENESES X,et al.Circulating miR-141-3p,miR-143-3p and miR-200c-3p are differentially expressed in colorectal cancer and advanced adenomas[J].Mol Clin Oncol,2019,11(2):201-207.
[18]	KONNO M,KOSEKI J,ASAI A,et al.Distinct methy-lation levels of mature microRNAs in gastrointestinal cancers[J].Nat Commun,2019,10(1):3888.
[19]	ZHANG Y,HUANG H F,ZHANG Y,et al.Combined detection of serum MiR-221-3p and MiR-122-5p expression in diagnosis and prognosis of gastric cancer[J].J Gastric Cancer,2019,19(3):315-328.
[20]	CHEN H,YANG Y Q,WANG J,et al.MiR-130b-5p promotes proliferation,migration and invasion of gastric cancer cells via targeting RASAL1[J].Oncol Lett,2018,15(5):6361-6367.
[21]	HANG C,YAN H S,GONG C,et al.MicroRNA-9 inhibits gastric cancer cell proliferation and migration by targeting neuropilin-1[J].Exp Ther Med,2019,18(4):2524-2530.
[22]	TAHARA T,TAHARA S,HORIGUCHI N,et al.Gastric mucosal microarchitectures associated with irreversibility with Helicobacter pylori eradication and downregulation of Micro RNA (miR)-124a[J].Cancer Invest,2019,37(9):417-426.
[23]	SUN C M,WU J,ZHANG H,et al.Circulating miR-125a but not miR-125b is decreased in active disease status and negatively correlates with disease severity as well as inflammatory cytokines in patients with Crohn's disease[J].World J Gastroenterol,2017,23(44):7888-7898.
[24]	REN H X,ZHANG F C,LUO H S,et al.Role of mast cell-miR-490-5p in irritable bowel syndrome[J].World J Gastroenterol,2017,23(1):93-102.
[25]	YOUNGER S T,COREY D R.Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters[J].Nucleic Acids Res,2011,39(13):5682-5691.
[26]	KREUZER-REDMER S,BEKURTZ J C,ARENDS D,et al.Feeding of Enterococcus faecium NCIMB 10415 leads to intestinal miRNA-423-5p-induced regulation of immune-relevant genes[J].Appl Environ Microbiol,2016,82(8):2263-2269.
[27]	LAMPROPOULOU D I,ARAVANTINOS G,LASCHOS K,et al.MiR-218 and miR-100 polymorphisms as markers of irinotecan-based chemotherapy response in metastatic colorectal cancer[J].Int J Colorectal Dis,2019,34(11):1871-1877.
[28]	HE Z Y,DANG J,SONG A L,et al.The involvement of miR-150/β-catenin axis in colorectal cancer progression[J].Biomed Pharmacother,2020,121:109495.
[29]	KWON K H,OHIGASHI H,MURAKAMI A.Dextran sulfate sodium enhances interleukin-1β release via activation of p38 MAPK and ERK1/2 pathways in murine peritoneal macrophages[J].Life Sci,2007,81(5):362-371.
[30]	WAETZIG G H,SEEGERT D,ROSENSTIEL P,et al.P38 mitogen-activated protein kinase is activated and linked to TNF-α signaling in inflammatory bowel disease[J].J.Immunol,2002,168(10):5342-5351.
[31]	NAGAE K,UCHI H,MORINO-KOGA S,et al.Glucagon-like peptide-1 analogue liraglutide facilitates wound healing by activating PI3K/Akt pathway in keratinocytes[J].Diabetes Res Clin Pract,2018,146:155-161.
[32]	LING L,GU S H,CHENG Y,et al.bFGF promotes Sca-1+ cardiac stem cell migration through activation of the PI3K/Akt pathway[J].Mol Med Rep,2018,17(2):2349-2356.
[33]	LV C,YANG S W,CHEN X,et al.MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway[J].J Clin Neurosci,2017,46:156-162.
[34]	GAO K,LIU L,DOU X X,et al.Doses Lactobacillus reuteri depend on adhesive ability to modulate the intestinal immune response and metabolism in mice challenged with lipopolysaccharide[J].Sci Rep,2016,6:28332.
[35]	GRIET M,ZELAYA H,MATEOS M V,et al.Soluble factors from Lactobacillus reuteri CRL1098 have anti-inflammatory effects in acute lung injury induced by lipopolysaccharide in mice[J].PLoS One,2014,9(10):e110027.
[36]	XU Y X,ZHANG J Z,FAN L,et al.miR-423-5p suppresses high-glucose-induced podocyte injury by targeting Nox4[J].Biochem Biophys Res Commun,2018,505(2):339-345.
[37]	XU L,SUN H B,XU Z N,et al.MicroRNA-218 regulates the epithelial-to-mesenchymal transition and the PI3K/Akt signaling pathway to suppress lung adenocarcinoma progression by directly targeting BMI-1[J].Eur Rev Med Pharmacol Sci,2019,23(18):7978-7988.
[38]	YANG B L,GE Y,ZHOU Y,et al.MiR-124a inhibits the proliferation and inflammation in rheumatoid arthritis fibroblast-like synoviocytes via targeting PIK3/NF-κB pathway[J].Cell Biochem Funct,2019,37(4):208-215.