ETEC感染猪小肠上皮细胞初期差异表达cricRNA的筛选

doi:10.16431/j.cnki.1671-7236.2023.09.005

摘要/Abstract

摘要： 【目的】分析产肠毒素大肠杆菌(enterotoxigenic Escherichia coli,ETEC)感染猪小肠上皮细胞(IPEC-J2)初期宿主细胞内环状RNA(circularRNA,circRNA)的表达谱变化,并探究其调控机制。【方法】采用Illumina PE150测序平台对ETEC感染IPEC-J2细胞进行转录组测序,利用生物信息学在线软件筛选出差异表达circRNAs,经过GO功能和KEGG通路富集分析筛选出ETEC感染相关circRNAs,用IRESfinder和PFAM 33.1软件进行circRNAs编码潜能预测;随机挑选5个差异表达circRNAs,用实时荧光定量PCR验证circRNAs表达情况及核糖核酸酶R(RNase R)消化结果。【结果】转录组测序结果显示,共获得201个差异表达circRNAs,其中95个表达上调和106个表达下调。GO功能富集分析表明,ETEC感染初期circRNAs来源基因主要影响细胞形态、细胞代谢和泛素蛋白连接酶活性等功能。KEGG通路富集分析显示,circRNAs可能参与氨基酸代谢途径、泛素介导的蛋白质水解通路、黏合连接和肌动蛋白细胞骨架调节等信号通路。circRNAs编码潜能预测发现4个具有编码潜能的circRNAs,其中novel_circ_0009996和来源基因TNFAIP3共同富集在NF-κB和TNF信号通路。实时荧光定量PCR结果显示,5个circRNAs的表达水平与测序结果一致,差异表达circRNAs均为环状RNA分子,测序结果准确可靠。【结论】ETEC感染IPEC-J2细胞初期差异表达circRNAs和其来源基因主要参与炎症反应、基因表达的转录后调控、细胞免疫过程、细胞骨架、细胞增殖和凋亡等相关生物过程,其中novel_circ_0009996具有编码潜能,主要富集在NF-κB和TNF信号通路。研究结果为深入探究circRNA在ETEC感染初期的调控机制提供理论基础。

关键词: 产肠毒素大肠杆菌(ETEC); 猪小肠上皮细胞; circRNA; 感染

Abstract: 【Objective】 The purpose of this experiment was to analyze the changes of circularRNA (circRNA) expression profile in host cells of porcine small intestinal epithelial cells (IPEC-J2) at the beginning of enterotoxigenic Escherichia coli (ETEC) infection,and explore the regulation mechanism of circRNA.【Method】 Transcriptome sequencing was performed on the ETEC infected IPEC-J2 cells using Illumina PE150 sequencing platform,and differentially expressed circRNAs were screened using bioinformatics online software.After GO function and KEGG pathway enrichment analysis,circRNAs associated with ETEC infection were screened.Coding potential of circRNAs was predicted by IRESfinder and PFAM 33.1 software.Five differentially expressed circRNAs were randomly selected,and the expression of circRNAs and the digestion of RNase R were verified by Real-time quantitative PCR.【Result】Transcriptome sequencing results showed that 201 differentially expressed circRNAs were obtained,of which 95 were up-regulated and 106 were down-regulated.GO functional enrichment analysis showed that circRNA-derived genes at the early stage of ETEC infection mainly affected cell morphology,cell metabolism and ubiquitin-protein ligase activit.KEGG pathway enrichment analysis indicated that circRNAs might be involved in amino acid metabolism pathway,ubiquitin-mediated proteolysis pathway,adhesion junction and actin cytoskeleton regulation.Four circRNAs with coding potential were identified,among which novel_circ_0009996 and source gene TNFAIP3 were co-enriched in NF-κB and TNF signaling pathways.Real-time quantitative PCR results showed that the expression of the 5 circRNAs were consistent with the sequencing results,and the differentially expressed circRNAs were all circular,and the sequencing results were accurate and reliable.【Conclusion】 circRNAs and the source genes were mainly involved in inflammatory response,post-transcriptional regulation of gene expression,cellular immunity,cytoskeleton,cell proliferation,apoptosis and other related biological processes.novel_circ_0009996 had coding potential,which was mainly concentrated in the NF-κB and TNF signaling pathways.The results provided a theoretical basis for further exploring the regulatory mechanism of circRNA in the early stage ETEC infection.

Key words: enterotoxigenic Escherichia coli(ETEC); porcine small intestinal epithelial cells; circRNA; infection

中图分类号:

S814.8

朱凯晴, 朱悦, 刘迎迎, 崔亚男, 蒋秉妤, 李妍. ETEC感染猪小肠上皮细胞初期差异表达cricRNA的筛选[J]. 中国畜牧兽医, 2023, 50(9): 3490-3498.

ZHU Kaiqing, ZHU Yue, LIU Yingying, CUI Yanan, JIANG Bingyu, LI Yan. Screening of Differentially Expressed cricRNA of Porcine Small Intestinal Epithelial Cells at Early ETEC infection[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(9): 3490-3498.

参考文献

[1] DUBREUIL J D,ISAACSON R E,SCHIFFERLI D M.Animal enterotoxigenic Escherichia coli[J].EcoSal Plus,2016,7(1):10.
[2] 蒋磊,周明旭,夏芃芃,等.产肠毒素大肠杆菌感染的分子致病机制[J].中国兽医学报,2014,34(9):1551-1560. JIANG L,ZHOU M X,XIA P P,et al.Molecular pathogenic mechanisms of enterotoxigenic Escherichia coli infection[J]. Chinese Journal of Veterinary Science,2014,34(9):1551-1560.(in Chinese)
[3] 黄登桂,周加义,高春起,等.产肠毒素大肠杆菌对仔猪肠道黏膜屏障功能的影响及其损伤修复研究进展[J].动物营养学报,2019,31(1):48-56. HUANG D G,ZHOU J Y,GAO C Q,et al.Research progress of effects of enterotoxigenic Escherichia coli on intestinal mucosal barrier function and injury repair of piglets[J]. Chinese Journal of Animal Nutrition,2019,31(1):48-56.(in Chinese)
[4] AMEZCUA R,FRIENDSHIP R M,DEWEY C E,et al.Presentation of postweaning Escherichia coli diarrhea in southern Ontario,prevalence of hemolytic E.coli serogroups involved,and their antimicrobial resistance patterns[J].Canadian Journal of Veterinary Research,2002,66(2):73-78.
[5] 严杜建.大肠杆菌对抗生素的耐药性研究进展[J].甘肃畜牧兽医,2013,43(3):26-28. YAN D J.Advances in the study of antibiotic resistance in Escherichia coli[J].Gansu Animal and Veterinary Sciences,2013,43(3):26-28.(in Chinese)
[6] 赵浩飞,赵红,徐永平,等.猪源产肠毒素大肠杆菌疫苗的研究进展[J].畜牧与兽医,2021,53(11):141-144. ZHAO H F,ZHAO H,XU Y P,et al.Research progress on the vaccines against enterotoxigenic Escherichia coli in swine industry[J].Animal Husbandry & Veterinary Medicine,2021,53(11):141-144.(in Chinese)
[7] AUGUSTINO S,XU Q,LIU X,et al.Transcriptomic study of porcine small intestine epithelial cells reveals important genes and pathways associated with susceptibility to Escherichia coli F4ac diarrhea[J].Frontiers in Genetics,2020,11:68.
[8] CHEN L,WANG C,SUN H,et al.The bioinformatics toolbox for circRNA discovery and analysis[J].Briefings in Bioinformatics,2021,22(2):1706-1728.
[9] LIU S,ZHANG D,LIU Y,et al.Circular RNA circ_0001105 protects the intestinal barrier of septic rats by inhibiting inflammation and oxidative damage and YAP1 expression[J].Gene,2020,755:144897.
[10] LI Z X,WANG L X,ZHANG Y,et al.circGLI3 inhibits oxidative stress by regulating the miR-339-5p/VEGFA axis in IPEC-J2 cells[J].BioMed Research International,2021,2021:1086206.
[11] CHEN J,WANG H,JIN L,et al.Profile analysis of circRNAs induced by Porcine endemic diarrhea virus infection in porcine intestinal epithelial cells[J].Virology,2019,527:169-179.
[12] 黄静.ETEC感染对猪肠道上皮细胞的炎性损伤作用及甘草多糖的干预效果研究[D].重庆:西南大学,2020. HUANG J.Effect of ETEC infection on inflammatory injury of porcine intestinal epithelial cells and the intervention effect of glycyrrhiza polysaccharide[D].Chongqing:Southwestern University,2020.(in Chinese)
[13] LIU M,ZHANG Y,ZHANG D,et al.Immunogenicity and protective efficacy of enterotoxigenic Escherichia coli (ETEC) total RNA against ETEC challenge in a mouse model[J].Scientific Reports,2020,10(1):20530.
[14] CHEN W,LV X,ZHANG W,et al.Non-coding transcriptome provides novel insights into the Escherichia coli F17 susceptibility of sheep lamb[J].Biology (Basel),2022,11(3):348.
[15] 聂晶,田春艳,张令强.泛素蛋白连接酶MDM2活性及稳定性调控的研究进展[J].遗传,2009,31(10):993-998. NEI J,TIAN C Y,ZHANG L Q.Progress in regulation of activity and stability of ubiquitin protein ligase MDM2[J].Hereditas,2009,31(10):993-998.(in Chinese)
[16] FUJITA Y,TINOCO R,LI Y,et al.Ubiquitin ligases in cancer immunotherapy-balancing antitumor and autoimmunity[J].Trends in Molecular Medicine,2019,25(5):428-443.
[17] SMITH J A.Regulation of cytokine production by the unfolded protein response;Implications for infection and autoimmunity[J].Frontiers in Immunology,2018,9:422.
[18] LAM M,MARSTERS S A,ASHKENAZI A,et al.Misfolded proteins bind and activate death receptor 5 to trigger apoptosis during unresolved endoplasmic reticulum stress[J].eLife,2020,9:e52291.
[19] XIA Y,BIN P,LIU S,et al.Enterotoxigenic Escherichia coli infection promotes apoptosis in piglets[J].Microbial Pathogenesis,2018,125:290-294.
[20] 罗钧秋,陈代文.赖氨酸对蛋白质代谢的影响及其可能调控机制[J].饲料工业,2006,16:40-43. LUO J Q,CHEN D W.Effects of lysine on protein metabolism and its possible regulatory mechanisms[J].Feed Industry,2006,16:40-43.(in Chinese)
[21] DANG F,NIE L,WEI W.Ubiquitin signaling in cell cycle control and tumorigenesis[J].Cell Death and Differentiation,2021,28(2):427-438.
[22] BALDWIN M R,BARBIERI J T.The type Ⅲ cytotoxins of Yersinia and Pseudomonas aeruginosa that modulate the actin cytoskeleton[J].Current Topics in Microbiology and Immunology,2005,291:147-166.
[23] NAVARRO-GARCIA F,SERAPIO-PALACIOS A,UGALDE-SILVA P,et al.Actin cytoskeleton manipulation by effector proteins secreted by diarrheagenic Escherichia coli pathotypes[J].BioMed Research International,2013,2013:374395.
[24] ZHANG M,HUANG N,YANG X,et al.A novel protein encoded by the circular form of the SHPRH gene suppresses glioma tumorigenesis[J].Oncogene,2018,37(13):1805-1814.
[25] ZHANG M,ZHAO K,XU X,et al.A peptide encoded by circular form of LINC-PINT suppresses oncogenic transcriptional elongation in glioblastoma[J].Nature Communications,2018,9(1):4475.
[26] ZHANG Q,YU K,CAO Y,et al.miR-125b promotes the NF-kappaB-mediated inflammatory response in NAFLD via directly targeting TNFAIP3[J].Life Sciences,2021,270:119071.
[27] WANG X,GAO X,HARDWIDGE P R.Heat-labile enterotoxin-induced activation of NF-kappaB and MAPK pathways in intestinal epithelial cells impacts enterotoxigenic Escherichia coli (ETEC) adherence[J].Microbial Cell Factories,2012,14(8):1231-1241.