C型产气荚膜梭菌实验感染仔猪肠道circRNA的表达特征

doi:10.11843/j.issn.0366-6964.2022.11.031

摘要/Abstract

摘要： circRNAs在病原菌感染宿主的肠道疾病发展中具有重要的调控作用。C型产气荚膜梭菌（C.perfringens type C）是引起仔猪腹泻及相关肠道炎症的主要细菌之一，对产业造成了严重的经济损失。然而，目前有关circRNAs如何调控仔猪C型产气荚膜梭菌性腹泻的全面且系统的研究未见报道。本研究通过RNA高通量测序研究分析了C型产气荚膜梭菌感染的7日龄仔猪回肠组织circRNAs的表达谱，筛选差异表达circRNAs，并进行差异表达基因GO和KEGG功能富集分析，利用miRanda等软件预测circRNA的microRNA靶点，构建circRNA-miRNA-mRNA的互作网络，并进行qPCR验证。结果显示，在C型产气荚膜梭菌感染的处理组（TI组）和对照组（CI组）中共鉴定出3 162个circRNAs，其中差异表达circRNAs有694个，上调表达circRNAs有404个，下调表达circRNAs有290个。功能富集分析结果表明，差异表达circRNAs的亲本基因主要富集在细胞周期、TGF-β信号通路、赖氨酸降解、Wnt信号通路、T细胞受体信号通路、MAPK信号通路等，调节仔猪对产气荚膜梭菌感染的抗性反应。此外，本研究构建了与C型产气荚膜梭菌感染致仔猪腹泻相关的circRNA-miRNA-mRNA互作网络，发现8 circRNAs-5 miRNAs-12 mRNAs组成的ceRNA网络与产气荚膜梭菌感染性疾病密切相关。本试验可为深入研究circRNA调控仔猪C型产气荚膜梭菌性腹泻疾病及猪抗腹泻病品系培育提供参考。

关键词: 仔猪腹泻, C型产气荚膜梭菌, circRNA, 炎性反应, 抗性

Abstract: circRNAs have important regulatory roles in enteric disease development of host infected by pathogenic bacteria. Clostridium perfringens type C (C. perfringens type C) is one of primary bacteria leading to a series of diarrhea and inflammatory diseases in piglets, which caused serious economic losses over the industry. However, the comprehensive and systematic understanding of circRNAs regulating piglet diarrhea caused by C. perfringens type C has not been reported. To illustrate the dynamics of piglet after C. perfringens infection, this study has investigated the expression profiles of ileum circRNAs of 7-day-old piglets challenged by C. perfringens type C through RNA sequencing, screened the differentially expressed circRNAs, and performed the GO and KEGG functional enriched functions. The targets of circRNAs were predicted by miRanda software, then constructing the circRNA/miRNA/mRNA interaction network and finally conducting the qPCR verification. A total of 3 162 circRNAs were identified with quantitative 694 differentially expressed circRNAs in treatment group(TI) and control group(CI) through sequencing, while 404 of which were up-regulated and 290 were down-regulated. Function analysis revealed that these dysregulated circRNAs were mostly enriched in several KEGG pathways including cell cycle, TGF-beta signaling pathway, Lysine degradation, Wnt signaling pathway, T cell receptor signaling pathway, MAPK signaling pathway and so on, regulating piglets resistance response to C. perfringens infection. Furthermore, the interactive network of circRNA-miRNA-mRNA were built in the C. perfringens type C-infected piglet ileum tissues, and the circRNA-associated ceRNA networks composed of 8 circRNAs-5 miRNAs-12 mRNAs was predicted to have important association with CAED. This study may provide valuable information for understanding the regulatory functions of circRNAs of piglet defensed to C. perfringens type C.

Key words: piglet diarrhea, Clostridium perfringenes type C, circRNA, inflammatory response, resistance

中图分类号:

S852.616.3

黄晓宇, 杨巧丽, 闫尊强, 王鹏飞, 石海仁, 滚双宝. C型产气荚膜梭菌实验感染仔猪肠道circRNA的表达特征[J]. 畜牧兽医学报, 2022, 53(11): 4058-4070.

HUANG Xiaoyu, YANG Qiaoli, YAN Zunqiang, WANG Pengfei, SHI Hairen, GUN Shuangbao. Characterization of circRNA Expression Profiles Involved in Intestines of Clostridium Perfringens Type C-infected Diarrheal Piglet[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4058-4070.

参考文献 48

[1]	ROOD J I.Virulence genes of Clostridium perfringens[J].Annu Rev Microbiol, 1998, 52:333-360.
[2]	ROOD J I, COLE S T.Molecular genetics and pathogenesis of Clostridium perfringens[J].Microbiol Rev, 1991, 55(4):621-648.
[3]	POPOFF M R, BOUVET P.Genetic characteristics of toxigenic Clostridia and toxin gene evolution[J].Toxicon, 2013, 75:63-89.
[4]	UZAL F A, VIDAL J E, MCCLANE B A, et al.Clostridium perfringens toxins involved in Mammalian veterinary diseases[J].Open Toxinology J, 2010, 2:24-42.
[5]	SONGER J G.Clostridia as agents of zoonotic disease[J].Vet Microbiol, 2010, 140(3-4):399-404.
[6]	SONGER J G, UZAL F A.Clostridial enteric infections in pigs[J].J Vet Diagn Invest, 2005, 17(6):528-536.
[7]	UZAL F A, MCCLANE B A.Recent progress in understanding the pathogenesis of Clostridium perfringens type C infections[J].Vet Microbiol, 2011, 153(1-2):37-43.
[8]	UZAL F A, MCCLANE B A, CHEUNG J K, et al.Animal models to study the pathogenesis of human and animal Clostridium perfringens infections[J].Vet Microbiol, 2015, 179(1-2):23-33.
[9]	POPESCU F, WYDER M, GURTNER C, et al.Susceptibility of primary human endothelial cells to C.perfringens beta-toxin suggesting similar pathogenesis in human and porcine necrotizing enteritis[J].Vet Microbiol, 2011, 153(1-2):173-177.
[10]	SILVA R O S, JUNIOR C A O, GUEDES R M C, et al.Clostridium perfringens:A review of the disease in pigs, horses and broiler chickens[J].Ciência Rural, 2015, 45(6):1027-1034.
[11]	DINH H, HONG Y H, LILLEHOJ H S.Modulation of microRNAs in two genetically disparate chicken lines showing different necrotic enteritis disease susceptibility[J].Vet Immunol Immunopathol, 2014, 159(1-2):74-82.
[12]	MA M L, GURJAR A, THEORET J R, et al.Synergistic effects of Clostridium perfringens enterotoxin and beta toxin in rabbit small intestinal loops[J].Infect Immun, 2014, 82(7):2958-2970.
[13]	UZAL F A, SAPUTO J, SAYEED S, et al.Development and application of new mouse models to study the pathogenesis of Clostridium perfringens type C enterotoxemias[J].Infect Immun, 2009, 77(12):5291-5299.
[14]	MEMCZAK S, JENS M, ELEFSINIOTI A, et al.Circular RNAs are a large class of animal RNAs with regulatory potency[J].Nature, 2013, 495(7441):333-338.
[15]	HANSEN T B, JENSEN T I, CLAUSEN B H, et al.Natural RNA circles function as efficient microRNA sponges[J].Nature, 2013, 495(7441):384-388.
[16]	JECK W R, SORRENTINO J A, WANG K, et al.Circular RNAs are abundant, conserved, and associated with alu repeats[J].RNA, 2013, 19(2):141-157.
[17]	GRUNER H, CORTÉS-LÓPEZ M, COOPER D A, et al.CircRNA accumulation in the aging mouse brain[J].Sci Rep, 2016, 6:38907.
[18]	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].Sci Rep, 2019, 9(1):1963.
[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].Res Vet Sci, 1990, 49(2):223-228.
[20]	LANGMEAD B, TRAPNELL C, POP M, et al.Ultrafast and memory-efficient alignment of short DNA sequences to the human genome[J].Genome Biol, 2009, 10(3):R25.
[21]	GLAŽAR P, PAPAVASILEIOU P, RAJEWSKY N.circBase:A database for circular RNAs[J].RNA, 2014, 20(11):1666-1670.
[22]	GAO Y, ZHANG J Y, ZHAO F Q.Circular RNA identification based on multiple seed matching[J].Brief Bioinform, 2018, 19(5):803-810.
[23]	ZHOU L, CHEN J H, LI Z Z, et al.Integrated profiling of microRNAs and mRNAs:microRNAs located on Xq27.3 associate with clear cell renal cell carcinoma[J].PLoS One, 2010, 5(12):e15224.
[24]	LOVE M I, HUBER W, ANDERS S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[J].Genome Biol, 2014, 15(12):550.
[25]	YOUNG M D, WAKEFIELD M J, SMYTH G K, et al.Gene ontology analysis for RNA-seq:accounting for selection bias[J].Genome Biol, 2010, 11(2):R14.
[26]	XIE C, MAO X Z, HUANG J J, et al.KOBAS 2.0:A web server for annotation and identification of enriched pathways and diseases[J].Nucleic Acids Res, 2011, 39(Web Server issue):W316-W322.
[27]	MAO X Z, CAI T, OLYARCHUK J G, et al.Automated genome annotation and pathway identification using the KEGG Orthology (KO) as a controlled vocabulary[J].Bioinformatics, 2005, 21(19):3787-3793.
[28]	SHANNON P, MARKIEL A, OZIER O, et al.Cytoscape:A software environment for integrated models of biomolecular interaction networks[J].Genome Res, 2003, 13(11):2498-2504.
[29]	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].Microb Pathog, 2019, 136:103699.
[30]	HUANG X Y, SUN W Y, YAN Z Q, et al.Integrative Analyses of long non-coding RNA and mRNA involved in piglet ileum immune response to Clostridium perfringens type C infection[J].Front Cell Infect Microbiol, 2019, 9:130.
[31]	LIU C X, LI X, NAN F, et al.Structure and degradation of circular RNAs regulate PKR activation in innate immunity[J].Cell, 2019, 177:865-880.
[32]	ZHENG Q P, BAO C Y, GUO W J, et al.Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs[J].Nat Commun, 2016, 7:11215.
[33]	EBBESEN K K, KJEMS J, HANSEN T B.Circular RNAs:Identification, biogenesis and function[J].Biochim Biophys Acta, 2016, 1859(1):163-168.
[34]	XIE H J, REN X L, XIN S N, et al.Emerging roles of circRNA_001569 targeting miR-145 in the proliferation and invasion of colorectal cancer[J].Oncotarget, 2016, 7(18):26680-26691.
[35]	EL-DALY S M, MORSY S M, MEDHAT D, et al.The diagnostic efficacy of circulating miRNAs in monitoring the early development of colitis-induced colorectal cancer[J].J Cell Biochem, 2019, 120(10):16668-16680.
[36]	DO D N, DUDEMAINE P L, FOMENKY B E, et al.Integration of miRNA and mRNA co-expression reveals potential regulatory roles of miRNAs in developmental and immunological processes in calf ileum during early growth[J].Cells, 2018, 7(9):134.
[37]	LIANG G X, MALMUTHUGE N, MCFADDEN T B, et al.Potential regulatory role of microRNAs in the development of bovine gastrointestinal tract during early life[J].PLoS One, 2014, 9(3):e92592.
[38]	XU Z, ZHANG Y Y, DING J J, et al.miR-17-3p downregulates mitochondrial antioxidant enzymes and enhances the radiosensitivity of prostate cancer cells[J].Mol Ther Nucleic Acids, 2018, 13:64-77.
[39]	KERKHOFF C, NACKEN W, BENEDYK M, et al.The arachidonic acid-binding protein S100A8/A9 promotes NADPH oxidase activation by interaction with p67phox and Rac-2[J].FASEB J, 2005, 19(3):467-469.
[40]	VOGL T, TENBROCK K, LUDWIG S, et al.MRP8 and MRP14 are endogenous activators of toll-like receptor 4, promoting lethal, endotoxin-induced shock[J].Nat Med, 2007, 13(9):1042-1049.
[41]	ZHAO B Y, LU R F, CHEN J J, et al.S100A9 blockade prevents lipopolysaccharide-induced lung injury via suppressing the NLRP3 pathway[J].Respir Res, 2021, 22(1):45.
[42]	ZHANG X M, WEI L Y, WANG J, et al.Suppression colitis and colitis-associated colon cancer by anti-S100A9 antibody in mice[J].Front Immunol, 2017, 8:1774.
[43]	CHEN L S, HUANG Y, DUAN Z X, et al.Exosomal miR-500 derived from lipopolysaccharide-treated macrophage accelerates liver fibrosis by suppressing MFN2[J].Front Cell Dev Biol, 2021, 9:716209.
[44]	ZHANG L, DING Y, YUAN Z Y, et al.MicroRNA-500 sustains nuclear factor-κB activation and induces gastric cancer cell proliferation and resistance to apoptosis[J].Oncotarget, 2015, 6(4):2483-2495.
[45]	DING P P, LI L, LI L Y, et al.C5aR1 is a master regulator in colorectal tumorigenesis via immune modulation[J].Theranostics, 2020, 10(19):8619-8632.
[46]	BLANCHARD H, LEGRAND P, PÉDRONO F.Fatty Acid Desaturase 3 (Fads3) is a singular member of the Fads cluster[J].Biochimie, 2011, 93(1):87-90.
[47]	JIN Y Y, WANG J, ZHANG M, et al.Role of bta-miR-204 in the regulation of adipocyte proliferation, differentiation, and apoptosis[J].J Cell Physiol, 2019, 234(7):11037-11046.
[48]	WANG W, YANG Q L, HUANG X Y, et al.Effects of miR-204 on apoptosis and inflammatory response of Clostridium perfringens beta2 toxin induced IPEC-J2 cells via targeting BCL2L2[J].Microb Pathog, 2021, 156:104906.