香猪ENTPD1基因3'UTR的SINE插入下调其基因表达

doi:10.11843/j.issn.0366-6964.2025.09.015

摘要/Abstract

摘要： 旨在探究香猪ENTPD1基因3'UTR短散在元件(short interspersed nuclear element, SINE)插入/缺失的群体多态性及对基因表达的影响。本研究以香猪为研究对象，以大白猪为对照，利用PCR技术检测香猪和大白猪群体中ENTPD1基因3'UTR区SINE插入/缺失的群体多态性；利用UCSC、SINE Base、miRNA Base、PITA及RBP suite等数据库及软件分析SINE序列中的功能元件；利用Real-time fluorescence quantitative PCR(RT-qPCR)技术检测6月龄健康香猪不同组织(心、肝、脾、肺、肾)ENTPD1的mRNA水平和不同基因型个体肺组织ENTPD1的mRNA水平，利用Western blotting技术检测不同基因型个体肺组织ENTPD1的蛋白水平。结果显示，香猪ENTPD1 3'UTR存在一个297 bp的SINE多态位点，位于终止密码子下游466 bp后，生物信息学分析发现，该SINE属于Pre0_SS tRNA家族，含有RNA聚合酶III启动子、多个RNA结合蛋白和miRNA的结合位点；在香猪和大白猪群体中均检测出3种基因型，即插入(SINE+/+)、缺失(SINE-/-)和杂合(SINE+/-)，香猪群体中缺失(SINE-/-)基因型频率极显著高于大白猪(P<0.01)，SINE-等位基因频率显著高于大白猪(P<0.05)，SINE插入/缺失在两个群体中均呈现中度多态性，但仅在香猪群体中符合Hardy-Weinberg平衡。香猪不同组织ENTPD1 mRNA检测显示脾、肺组织中mRNA水平较高。香猪不同基因型肺组织ENTPD1的mRNA和蛋白检测显示，SINE-/-、SINE+/-基因型的mRNA水平极显著高于SINE+/+，SINE-/-基因型蛋白水平极显著高于SINE+/-、SINE+/+。研究结果提示，SINE插入负调控ENTPD1基因的表达。

关键词: ENTPD1, 3'UTR, SINE, 基因表达, 香猪

Abstract: The study aimed to investigate the population polymorphism of short interspersed nuclear element (SINE) insertions/deletions in the 3'UTR of the ENTPD1 gene and their effect on gene expression in the Xiang pig. Xiang pigs were used as research subjects and Large White pigs were used as controls. The detection of population polymorphism of SINE insertions/deletions in the 3'UTR region of the ENTPD1 gene in Xiang pig and Large White pig populations was performed by PCR. The functional elements in SINE sequences was analyzed using databases and softwares such as UCSC, SINE Base, miRNA Base, PITA and RBP suite; The mRNA levels of ENTPD1 in different tissues (heart, liver, spleen, lung and kidney) of individuals of 6-month-old and mRNA levels of ENTPD1 in lung tissues of individuals with different genotypes of healthy adult Xiang pig was detected using Real-time fluorescence quantitative PCR (RT-qPCR) technology. The protein levels of ENTPD1 in lung tissues of individuals with different genotypes was detected using Western blotting technique. The results showed that there was a 297 bp SINE polymorphism site in the 3'UTR of ENTPD1 in Xiang pig, it was located at 466 bp downstream of the terminator codon. Bioinformatic analysis revealed that the SINE belonged to the Pre0_SS tRNA family and contained the RNA polymerase III promoter, multiple RNA-binding proteins, and binding sites for miRNAs; Three genotypes, insertion (SINE+/+), deletion (SINE-/-) and heterozygote (SINE+/-), were detected in Xiang pigs and Large White pigs. The frequency of deletion (SINE-/-) genotype in Xiang pigs was significantly higher than that in Large White pigs (P<0.01), and the frequency of SINE- allele was significantly higher than that in Large White pigs (P<0.05). The variation showed moderate polymorphism in both populations, but only in Xiang pig population conformed to Hardy-Weinberg equilibrium. Detection of ENTPD1 mRNA in different tissues of Xiang pig showed higher mRNA levels in spleen and lung tissues. The mRNA and protein assays of ENTPD1 in lung tissues of individuals with different genotypes of Xiang pigs showed that the mRNA levels of SINE-/- and SINE+/- genotypes were highly significantly higher than those of SINE+/+, and the protein levels of SINE-/- genotypes were highly significantly higher than those of SINE+/- and SINE+/+. The results suggest that SINE insertion negatively regulates the expression of ENTPD1 gene.

Key words: ENTPD1, 3'UTR, SINE, gene expression, Xiang pig

中图分类号:

S828.2

田姣, 龙菊烟, 陈霞, 岑晓丽, 牛熙, 黄世会, 王嘉福, 冉雪琴. 香猪ENTPD1基因3'UTR的SINE插入下调其基因表达[J]. 畜牧兽医学报, 2025, 56(9): 4303-4314.

TIAN Jiao, LONG Juyan, CHEN Xia, CEN Xiaoli, NIU Xi, HUANG Shihui, WANG Jiafu, RAN Xueqin. Down-regulation of Gene Expression by SINE Insertion in the 3'UTR of the ENTPD1 Gene in the Xiang Pig[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(9): 4303-4314.

参考文献

[1] LI X Y, MOESTA A K, XIAO C, et al. Targeting CD39 in cancer reveals an extracellular ATP- and inflammasome-driven tumor immunity[J]. Cancer Discov, 2019, 9(12): 1754-1773.
[2] SCHADLICH I S, WINZER R, STABERNACK J, et al. The role of the ATP-adenosine axis in ischemic stroke[J]. Semin Immunopathol, 2023, 45(3): 347-365.
[3] WANG C, YINQ, SN Z, et al. Progress on role of extracellular ATP and its metabolite adenosine in immunoregulation: Review[J]. Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2020, 36(12): 1134-1140.
[4] GRASSI F, MARINO R. The P2X7 receptor in mucosal adaptive immunity[J]. Purinergic Signal, 2024, 20(1): 9-19.
[5] VUERICH M, ROBSON S C, LONGHI M S. Ectonucleotidases in intestinal and hepatic inflammation[J]. Front Immunol, 2019, 10: 507.
[6] GUTKNECHT DA SILVA J L, PASSOS D F, CABRAL F L, et al. Istradefylline induces A2A/P2X7 crosstalk expression inducing pro-inflammatory signal, and reduces AKT/mTOR signaling in melanoma-bearing mice[J]. Med Oncol, 2023, 40(6): 178.
[7] PENG Z W, ROTHWEILER S, WEI G, et al. The ectonucleotidase ENTPD1/CD39 limits biliary injury and fibrosis in mouse models of sclerosing cholangitis[J]. Hepatol Commun, 2017, 1(9): 957-972.
[8] LEE N T, SAVVIDOU I, SELAN C, et al. Development of endothelial-targeted CD39 as a therapy for ischemic stroke[J]. J Thromb Haemost, 2024, 22(8): 2331-2344.
[9] ZENG J, NING Z, WANG Y, et al. Implications of CD39 in immune-related diseases[J]. Int Immunopharmacol, 2020, 89: 107055.
[10] SAVIO L E B, DE ANDRADE MELLO P, FIGLIUOLO V R, et al. CD39 limits P2X7 receptor inflammatory signaling a nd attenuates sepsis-induced liver injury[J]. J Hepatol, 2017, 67(4): 716-726.
[11] MALONEY J P, BRANCHFORD B R, BRODSKY G L, et al. The ENTPD1 promoter polymorphism -860 A>G (rs3814159) is associated with increased gene transcription, protein expression, CD39/NTPDase1 enzymatic activity, and thromboembolism risk[J]. Faseb j, 2017, 31(7): 2771-2784.
[12] ZHAO P, GU L, GAO Y, et al. Young SINEs in pig genomes impact gene regulation, genetic diversity, and complex traits[J]. Commun Biol, 2023, 6(1): 894.
[13] TAM P L F, LEUNG D. The molecular impacts of retrotransposons in development and diseases[J]. Int J Mol Sci, 2023, 24(22): 16418.
[14] ZHENG Y, CHEN C, WANG M, et al. SINE insertion in the pig carbonic anhydrase 5B (CA5B) gene is associated with changes in gene expression and phenotypic variation[J]. Animals (Basel), 2023, 13(12):1942.
[15] KRAMEROV D A, VASSETZKY N S. Origin and evolution of SINEs in eukaryotic genomes[J]. Heredity (Edinb), 2011, 107(6): 487-495.
[16] SENFT A D, MACFARLAN T S. Transposable elements shape the evolution of mammalian development[J]. Nat Rev Genet, 2021, 22(11): 691-711.
[17] CHOI J D, DEL PINTO L A, SUTTER N B. SINE retrotransposons import polyadenylation signals to 3'UTRs in dog (Canis familiaris)[J]. Mob DNA, 2025, 16(1): 1.
[18] WANG X, CHI C, HE J, et al. SINE insertion may act as a repressor to affect the expression of pig LEPROT and growth traits[J]. Genes (Basel), 2022, 13(8):1422.
[19] QI F, CHEN X, WANG J, et al. Genome-wide characterization of structure variations in the Xiang pig for genetic resistance to African swine fever[J]. Virulence, 2024, 15(1): 2382762.
[20] 梁小芬, 冉雪琴, 牛熙, 等. 香猪CYP3A29基因3'-UTR结构变异及其与肌肉雄激素水平的关联分析[J]. 中国畜牧兽医, 2023, 50(8): 3199-3209.
LIANG X F,RAN X Q,NIU X, et al.Analysis of 3'-UTR Structural variations of CYP3A29 gene in Xiang pigs and its association with muscle androgen levels[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(8): 3199-3209. (in Chinese)
[21] PENG Z W, ROTHWEILER S, WEI G, et al. The ectonucleotidase ENTPD1/CD39 limits biliary injury and fibrosis in mouse models of sclerosing cholangitis[J]. Hepatol Commun, 2017, 1(9): 957-972.
[22] SABATEL C, BUREAU F. The innate immune brakes of the lung[J]. Front Immunol, 2023, 14: 1111298.
[23] RYANTO G R T, SURAYA R, NAGANO T. The importance of lung innate immunity during health and disease[J]. Pathogens, 2025, 14(1):91.
[24] WANG L, LI Y J, YANG X, et al. Purinergic signaling: a potential therapeutic target for ischemic stroke[J]. Purinergic Signal, 2023, 19(1): 173-183.
[25] ROBSON S C, WU Y, SUN X, et al. Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation[J]. Semin Thromb Hemost, 2005, 31(2): 217-233.
[26] ORTIZ M A, DIAZ-TORNÉ C, DE AGUSTIN J J, et al. Altered CD39 and CD73 expression in rheumatoid arthritis: Implications for disease activity and treatment response[J]. Biomolecules, 2023, 14(1):1.
[27] 李默晗. CD39通过下调PAI-1表达与活性保护缺血性卒中脑组织[D]. 长沙:中南大学, 2022.
LI M H. CD39 protects the brain tissue of ischemic stroke by down-regulating the expression and activity of PAI-1[D]. Changsha: Zhongnan University, 2022. (in Chinese)
[28] 夏国庆. ATP通过P2X4和CD39调控酒精相关性脂肪性肝炎机制研究[D]. 合肥:安徽医科大学,2022.
XIA G Q. Research on the Mechanism of ATP regulating Alcoholic Steatohepatitis through P2X4 and CD39[D]. Hefie: Anhui Medical University, 2022. (in Chinese)
[29] 杜鹏. CD39-腺苷-A2B信号通路在低温携氧机械灌注改善DCD肝脏缺血再灌注损伤中的作用及机制研究[D]. 南昌:南昌大学, 2022.
DU P. Role and mechanism of CD39-adenosine-A2B signaling pathway in hypothermic oxygen-carrying mechanical perfusion to ameliorate liver ischemia-reperfusion injury in DCD[D]. Nanchang:Nanchang University, 2022. (in Chinese)
[30] LIU J J, RAN X Q, LI S, et al. Polymorphism in the first intron of follicle stimulating hormone beta gene in three Chinese pig breeds and two European pig breeds[J]. Anim Reprod Sci, 2009, 111(2-4): 369-375.
[31] MAGOTRA A, NASKAR S, DAS B, et al. A comparative study of SINE insertion together with a mutation in the first intron of follicle stimulating hormone beta gene in indigenous pigs of India[J]. Mol Biol Rep, 2015, 42(2): 465-470.
[32] 黄月丽, 冉雪琴, 牛熙, 等. 香猪CHD3基因3'-侧翼区264 bp结构变异的研究[J]. 中国畜牧兽医, 2022, 49(8): 3026-3035.
HUANG Y L, RAN X Q, NIU X,et al. A study of 264 bp structural variation in the 3'-flanking region of the CHD3 gene of the balsam pig[J]. China Animal Husbandry & Veterinary Medicine, 2022, 49(8): 3026-3035. (in Chinese)
[33] ZHAO P, GU L, GAO Y, et al. Young SINEs in pig genomes impact gene regulation, genetic diversity, and complex traits[J]. Commun Biol, 2023, 6(1): 894.
[34] TUCKER J M, GLAUNSINGER B A. Host noncoding retrotransposons induced by DNA viruses: a SINE of infection?[J]. J Virol, 2017, 91(23):e00982-17.
[35] KARIJOLICH J, ABERNATHY E, GLAUNSINGER B A. Infection-induced retrotransposon-derived noncoding RNAs enhance herpesviral gene expression via the NF-κB pathway[J]. PLoS Pathog, 2015, 11(11): e1005260.
[36] HWANG H, CHANG H R, BAEK D. Determinants of functional microRNA targeting[J]. Mol Cells, 2023, 46(1): 21-32.
[37] XI E, BAI J, ZHANG K, et al. Genomic variants disrupt miRNA-mRNA regulation[J]. Chem Biodivers, 2022, 19(10): e202200623.
[38] 李河林, 蒋玉芬, 程娜, 等. 筛选的差异表达microRNAs对猪卵母细胞Npm2基因的表达调控及作用研究[J]. 畜牧兽医学报, 2024, 55(11): 5035-5049.
LI H L, JIANG Y F, CHENG N, et al. Regulation of Npm2 gene expression and role of screened differentially expressed microRNAs in porcine oocytes[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(11): 5035-5049. (in Chinese)
[39] KOH D, BIN JEON H, OH C, et al. RNA-binding proteins in cellular senescence[J]. Mech Ageing Dev, 2023, 214: 111853.
[40] 余祖华, 高梦茹, 齐志颖, 等. RNA结合蛋白ELAVL1的功能及其调控病毒复制的研究进展[J]. 畜牧兽医学报, 2024, 55(5): 1914-1925.
YU Z H, GAO M R, QI Z Y, et al. Advances in the function of the RNA-binding protein ELAVL1 and its regulation of viral replication[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(5): 1914-1925. (in Chinese)
[41] AMEIS D, LIU F, KIRBY E, et al. The RNA-binding protein Quaking regulates multiciliated and basal cell abundance in the developing lung[J]. Am J Physiol Lung Cell Mol Physiol, 2021, 320(4): L557-L567.