感染肺炎支原体姜曲海猪肺组织差异表达circRNAs的筛选与分析

doi:10.16431/j.cnki.1671-7236.2021.06.035

摘要/Abstract

摘要： 为探明姜曲海猪感染猪肺炎支原体（Mycoplasma hyopneumoniae，Mhp）后肺组织环状RNA （circular RNA，circRNA）差异性表达谱及其在抗Mhp感染中的作用，试验以姜曲海猪为研究对象，分为感染组和对照组，人工感染Mhp 28 d后，解剖采集肺组织，采用高通量测序和生物信息学软件分析circRNA的表达情况。测序数据比对参考猪基因组序列，共鉴定到23 632个circRNAs，差异表达circRNAs为213个，其中97个上调，116个下调。随机选择4个差异表达circRNAs进行实时荧光定量PCR验证，检测结果与测序结果基本一致。差异表达circRNA来源基因可注释到包括抗原加工递呈、溶酶体、白细胞跨内皮迁移等免疫应答信号通路。circRNA-miRNA-mRNA靶标关系分析显示，筛选到海绵结合miRNA数量最多的3个差异表达circRNAs，分别为：circRNA-17284（21个）、circRNA-04848（19个）和circRNA-17270（19个）；预测到6个与免疫调控相关的靶向miRNAs，分别为：ssc-miR-4331、ssc-miR-370、ssc-miR-328、ssc-miR-30c-3p、ssc-miR-122和ssc-miR-125b。本研究测定了感染Mhp的猪肺组织circRNA表达谱，筛选到与免疫调控相关的差异表达circRNA，这有助于阐明姜曲海猪对Mhp的易感机制，为抗病育种研究提供了参考依据。

关键词: 姜曲海猪; 猪肺炎支原体; 环状RNA; 高通量测序

Abstract: In order to investigate the differential expression profile of circular RNA (circRNA) in the lung tissue of pigs infected with Mycoplasma hyopneumoniae (Mhp) as well as its role in Mhp infection.Jiangquhai pigs were used as subjects in this experiment,which were divided into Mhp-infected and control groups.On the 28th day post inoculation,the lung tissues were collected from all of the experimental pigs for analysis of circRNA expression by high-throughput sequencing and bioinformatics software.Data comparison showed that a total of 23 632 circRNAs were identified,with 213 differentially expressed circRNAs(DEcircRNAs).Among them,97 up-regulated and 116 down-regulated.Four from 213 DEcircRNAs were randomly selected and verified by Real-time quantitative PCR,and the results were consistent with those obtained by sequencing.Host genes of DEcircRNAs were annotated to immune-related signaling pathways,including antigen processing and presentation,lysosome and leukocyte transendothelial migration.The target relationship analysis of circRNA-miRNA-mRNA showed that three DEcircRNAs with the highest number of sponge miRNAs were circRNA-17284 (21),circRNA-04848 (19) and circRNA-17270 (19),of which 6 targeted miRNAs related to immune regulation were predicted,including ssc-miR-4331,ssc-miR-370,ssc-miR-328,ssc-miR-30c-3p,ssc-miR-122 and ssc-miR-125b.In this study,the expression profile of circRNA in the lung tissue of pigs infected with Mhp was determined,and the DEcircRNAs related to immune regulation was screened,which helped to clarify the mechanism of susceptibility of Jiangquhai pigs to Mhp infection,and provide scientific basis for disease-resistant breeding research.

Key words: Jiangquhai pigs; Mycoplasma hyopneumoniae; circRNA; high throughput sequencing

中图分类号:

Q786

倪黎纲, 赵旭庭, 王宵燕, 徐盼, 甘源, 金文. 感染肺炎支原体姜曲海猪肺组织差异表达circRNAs的筛选与分析[J]. 中国畜牧兽医, 2021, 48(6): 2212-2220.

NI Ligang, ZHAO Xuting, WANG Xiaoyan, XU Pan, GAN Yuan, JIN Wen. Screening and Analysis of Differentially Expressed circRNAs in the Lung Tissue of Jiangquhai pigs Infected with Mycoplasma hyopneumoniae[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(6): 2212-2220.

参考文献

[1] JECK W R,SHARPLESS N E.Detecting and characterizing circular RNAs[J].Nature Biotechnology,2014,32(5):453-461.
[2] 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.
[3] WANG K,LONG B,LIU F,et al.A circular RNA protects the heart from pathological hypertrophy and heart failure by targeting miR-223[J].European Heart Journal,2016,37(33):2602-2611.
[4] ZHENG Q,BAO C,GUO W,et al.Circular RNA profiling reveals an abundant circHIPK3 that regulates cell growth by sponging multiple miRNAs[J].Nature Communications,2016,7(11215):1-13.
[5] MAES D,SEGALES J,MEYNS T,et al.Control of Mycoplasma hyopneumoniae infections in pigs[J].Veterinary Microbiology,2008,126(4):297-309.
[6] 李彦伟,刘茂军,武昱孜,等.猪肺炎支原体致病机理研究进展[J].动物医学进展,2013,34(8):84-88. LI Y W,LIU M J,WU Y Z,et al.Advance in pathogenic mechanism of Mycoplasma hyopneumoniae[J].Progress in Veterinary Medicine,2013,34(8):84-88.(in Chinese)
[7] 沈青春,宁宜宝,覃青松.猪肺炎支原体的研究进展[J].中国兽药杂志,2003,37(6):26-30. SHEN Q C,NING Y B,QIN Q S.The progress of prevention and cure on swine Mycoplasma hyopneu-moniae[J].Chinese Journal of Veterinary Drug,2003,37(6):26-30.(in Chinese)
[8] 方晓敏,赵为民,付言峰,等.猪支原体肺炎发生的品种敏感差异及分子基础[J].中国农业科学,2015,48(14):2839-2847. FANG X M,ZHAO W M,FU Y F,et al.Difference in susceptibility to Mycoplasma pneumonia among various pig breeds and its molecular genetic basis[J].Scientia Agricultura Sinica,2015,48(14):2839-2847.(in Chinese)
[9] 邵国青,刘茂军,孙佩元,等.猪气喘病实验猪模型的建立[J].微生物与感染,2007,2(4):215-218. SHAO G Q,LIU M J,SUN P Y,et al.The establishment of an experimental swine model of swine Mycoplasma pneumonia[J].Journal of Microbes and Infection,2007,2(4):215-218.(in Chinese)
[10] MIRI D,SCHRAGA S,SARIT E,et al.Transcriptome-wide discovery of circular RNAs in archaea[J].Nucleic Acids Research,2012,40(7):3131-3142.
[11] LU T,CUI L,ZHOU Y,et al.Transcriptome-wide investigation of circular RNAs in rice[J].RNA,2015,21(12):2076-2087.
[12] ZHANG X,YAN Y,LEI X,et al.Circular RNA alterations are involved in resistance to Avian leukosis virus subgroup-J-induced tumor formation in chickens[J].Oncotarget,2017,8(21):34961-34970.
[13] 张晓,冉雪琴,牛熙,等.香猪3种组织环状RNA的表达及其功能分析[J].中国畜牧兽医,2020,47(12):86-97. ZHANG X,RAN X Q,NIU X,et al.Expression and function analysis of circular RNA in three tissues of Xiang pig[J].China Animal Husbandry & Veterinary Medicine,2020,47(12):86-97.(in Chinese)
[14] 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.
[15] MA X L,ZHAO X M,ZHANG Z C,et al.Differentially expressed non-coding RNAs induced by transmissible gastroenteritis virus potentially regulate inflammation and NF-κB pathway in porcine intestinal epithelial cell line[J].BMC Genomics,2018,19(1):747-760.
[16] CHEN J N,WANG H W,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.
[17] 倪黎纲,赵旭庭,王宵燕,等.基于高通量测序分析肺炎支原体感染姜曲海猪肺组织miRNA表达谱[J].浙江农业学报,2019,31(12):1979-1986. NI L G,ZHAO X T,WANG X Y,et al.microRNA sequencing and analysis of porcine lung of Jiangquhai pig response to Mycoplasma hyopneumoniae infection[J].Acta Agriculturae Zhejiangensis,2019,31(12):1979-1986.(in Chinese)
[18] SIBILA M,MENTABERRE G,BOADELLA M,et al.Serological,pathological and polymerase chain reaction studies on Mycoplasma hyopneumoniae infection in the wild boar[J].Veterinary Microbiology,2010,144(1):214-218.
[19] LI B,DU L P,SUN B,et al.Transcription analysis of the porcine alveolar macrophage response to Mycoplasma hyopneumoniae[J].PLoS One,2014,9(8):e101968.
[20] PAN Q,WANG X,LIU T,et al.Mycoplasma hyopneumoniae inhibits porcine beta-defensin 2 production by blocking the unfolded protein response to facilitate epithelial adhesion and infection[J].Infection and Immunity,2020,88(7):e00164-20.
[21] CHEN L L.The biogenesis and emerging roles of circular RNAs[J].Nature Reviews Molecular Cell Biology,2016,17(4):205-211.
[22] CHEN J,CUI L,YUAN J,et al.Circular RNA WDR77 target FGF-2 to regulate vascular smooth muscle cells proliferation and migration by sponging miR-124[J].Biochemical and Biophysical Research Communications,2017,494(1):126-132.
[23] XU X W,ZHENG B A,HU Z M,et al.Circular RNA hsa_circ_000984 promotes colon cancer growth and metastasis by sponging miR-106b[J].Oncotarget,2017,8(53):91674-91683.
[24] ZHAO X,BAI X,GUAN L,et al.microRNA-4331 promotes Transmissible gastroenteritis virus (TGEV)-induced mitochondrial damage via targeting RB1,upregulating interleukin-1 receptor accessory protein (IL1RAP),and activating p38 MAPK pathway in vitro[J].Molecular & Cellular Proteomics,2018,17(2):190-204.
[25] ZHANG S,WANG R,SU H,et al.Sus scrofa miR-204 and miR-4331 negatively regulate swine H1N1/2009 Influenza A virus replication by targeting viral HA and NS,respectively[J].International Journal of Molecular Sciences,2017,18(4):749-767.