香猪CTSV基因3'-UTR插入/缺失结构变异介导的基因表达差异研究

doi:10.16431/j.cnki.1671-7236.2025.11.021

摘要/Abstract

摘要： 【目的】组织蛋白酶V(cathepsins V，CTSV)是一种溶酶体半胱氨酸蛋白酶，在多种疾病中表达异常，而结构变异(structural variation，SV)是基因组多样性及基因表达差异的重要原因。试验旨在探究CTSV基因3'-UTR 517 bp插入/缺失SV在香猪群体中的遗传多样性及对基因表达的影响，为香猪的优良遗传选育提供潜在的分子标记。【方法】选取319头香猪作为试验动物，245头大白猪作为对照猪种，通过PCR扩增进行基因分型，分析CTSV基因3'-UTR SV在香猪和大白猪中的基因型分布情况及群体遗传特性；采用UCSC、miRanDa、PITA及RBPsuite等软件分析该SV包含的重复元件、miRNA结合位点和RNA结合蛋白(RNA binding protein，RBP)结合位点。应用实时荧光定量PCR检测CTSV基因不同基因型在香猪肺脏组织中的表达差异，并利用Western blotting技术检测CTSV蛋白表达水平。【结果】通过PCR基因分型检测，在香猪和大白猪中均检测到了野生型(WW)、杂合型(WD)、缺失型(DD)，且WW均为优势基因型，但香猪群体中缺失型(D)等位基因频率(32.29%)极显著高于大白猪D等位基因频率(8.57%)(P＜0.01)。经群体遗传特性分析，CTSV基因3'-UTR SV在香猪群体中为中度多态，在大白猪群体中为低度多态；χ²检验发现，该SV在2个猪群中均偏离Hardy-Weinberg平衡状态(P＜0.05)。生物信息学分析结果显示，香猪CTSV基因3'-UTR SV含有1个短散在元件(short interspersed nuclear element，SINE)重复序列、18个miRNA和22个RBP结合位点。实时荧光定量PCR和Western blotting结果显示，在香猪肺脏组织中CTSV基因WD和DD基因型的mRNA和蛋白表达量显著或极显著高于WW基因型(P＜0.05；P＜0.01)。【结论】香猪CTSV基因3'-UTR的517 bp缺失，导致该SV中含有SINE元件，miRNA和RBPs结合位点缺失或改变，并使CTSV基因异常表达和积累。

关键词: 香猪; CTSV基因; 结构变异; 多态性; 表达

Abstract: 【Objective】 Cathepsin V (CTSV),a lysosomal cysteine protease,exhibits abnormal expression in various diseases,and structural variation (SV) is a significant contributor to genomic diversity and differential gene expression.This study aimed to explore the genetic diversity and effect on gene expression of a 517 bp insertion/deletion SV in the 3'-UTR of CTSV gene in Xiang pig populations,providing potential molecular markers for the excellent genetic breeding of Xiang pigs. 【Method】 With 319 Xiang pigs as experimental subjects and 245 Large White pigs as control breeds,gene typing was conducted via PCR technology.The genotype distribution and genetic characteristic of the 3'-UTR SV of CTSV gene in Xiang pigs and Large White pigs was analyzed.Software such as UCSC,miRanDa,PITA,and RBPsuite were utilized to analyze the repetitive elements,miRNA binding sites,and RNA binding protein (RBP) binding sites contained within 3'-UTR SV of CTSV gene.Real-time quantitative PCR was applied to detect the expression differences of CTSV gene with different genotypes in lung of Xiang pigs.Meanwhile,Western blotting was used to detect the expression of CTSV protein. 【Result】 Through PCR genotyping,wild-type (WW),heterozygous (WD),and deletion-type (DD) were detected in both Xiang pigs and Large White pigs,with WW being the predominant genotype in both breeds.However,the allele frequency of the deletion-type (D) in Xiang pigs (32.29%) was extremely significantly higher than that in Large White pigs (8.57%)(P＜0.01).Population genetic characteristic analysis revealed that the 3'-UTR SV of CTSV gene exhibited moderate polymorphism in Xiang pigs and low polymorphism in Large White pigs.χ² test found that this SV was not in Hardy-Weinberg equilibrium in both pig populations (P＜0.05).Bioinformatics analysis showed that the 3'-UTR SV of CTSV gene contained one short interspersed nuclear element (SINE) repeat sequence,18 miRNA,and 22 RBP binding sites.Real-time quantitative PCR and Western blotting analysis results showed that the mRNA and protein expression of CTSV gene in lung of Xiang pigs with WD and DD genotypes were significantly or extremely significantly higher than those with WW genotype (P＜0.05 or P＜0.01). 【Conclusion】 The 517 bp deletion of CTSV gene 3'-UTR resulted in the presence of SINE elements in the SV,leading to the loss or alteration of binding sites for miRNA and RBPs,and causing abnormal expression and accumulation of CTSV gene.

Key words: Xiang pigs; CTSV gene; structural variation; polymorphism; expression

中图分类号:

S828

龙菊烟, 田姣, 岑晓丽, 陈霞, 牛熙, 黄世会, 王嘉福, 冉雪琴. 香猪CTSV基因3'-UTR插入/缺失结构变异介导的基因表达差异研究[J]. 中国畜牧兽医, 2025, 52(11): 5243-5253.

LONG Juyan, TIAN Jiao, CEN Xiaoli, CHEN Xia, NIU Xi, HUANG Shihui, WANG Jiafu, RAN Xueqin. Gene Differential Expression Mediated by Insertion/Deletion Structural Variants in 3'-UTR of CTSV Gene in Xiang Pigs[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(11): 5243-5253.

参考文献

[1] HÖLZEN L,PARIGIANI M A,REINHECKEL T.Tumor cell- and microenvironment-specific roles of cysteine cathepsins in mouse models of human cancers[J].Biochimica et Biophysica Acta-Proteins and Proteomics,2020,1868(7):140423.
[2] 侯昕彤,周慧.组织蛋白酶V的研究进展[J].沈阳医学院学报,2018,20(5):464-466.HOU X T,ZHOU H.Research progress of cathepsin V[J].Journal of Shenyang Medical College,2018,20(5):464-466.(in Chinese)
[3] LECAILLE F,CHAZEIRAT T,SAIDI A,et al.Cathepsin V:Molecular characteristics and significance in health and disease[J].Molecular Aspects of Medicine,2022,88:101086.
[4] LALMANACH G,SAIDI A,MARCHAND-ADAM S,et al.Cysteine cathepsins and cystatins:From ancillary tasks to prominent status in lung diseases[J].Biological Chemistry,2015,396(2):111-130.
[5] NAUMNIK W,OSSOLI AN＇G SKA M,PO AN＇G SKA I,et al.Endostatin and cathepsin-V in bronchoalveolar lavage fluid of patients with pulmonary sarcoidosis[J].Advances in Experimental Medicine and Biology,2015,833:55-61.
[6] ZHU L F,ZENG Q,WANG J X,et al.Cathepsin V drives lung cancer progression by shaping the immunosuppressive environment and adhesion molecules cleavage[J].Aging,2023,122:151-168.
[7] 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.Association analysis between structural variation of CYP3A29 gene 3'-UTR and androgen level of muscle tissue in Xiang pigs[J].China Animal Husbandry & Veterinary Medicine, 2023,50(8):3199-3209.(in Chinese)
[8] 胡江,王嘉福,牛熙,等.贵州地方猪种与其它猪种功能缺失相关基因的比较与分析[J].山地农业生物学报,2019,38(6):64-70.HU J,WANG J F,NIU X,et al.Comparison of releated genes to functional loss between Guizhou local pig breeds and other pig breeds[J].Journal of Mountain Agriculture and Biology,2019,38(6):64-70.(in Chinese)
[9] XU X L,YU Z G,AI N N,et al.Molecular mechanism of MYL4 regulation of skeletal muscle development in pigs[J].Genes (Basel),2023,14(6):1267.
[10] 贾紫森.NPC1基因突变对肾脏细胞粘附、增殖和凋亡的影响[D].新乡:新乡医学院,2023.JIA Z S.The effect of NPC1 gene mutation on adhesion,proliferation,and apoptosis of renal cells[D].Xinxiang:Xinxiang Medical University,2023.(in Chinese)
[11] NUGENT A A,LIN K,VAN L B,et al.TREM2 regulates microglial cholesterol metabolism upon chronic phagocytic challenge[J]. Neuron,2020,105(5):837-854.e9.
[12] 徐婷婷.基于CD基因结构变异建立香猪喘气病易感性检测技术[D].贵阳:贵州大学,2024.XU T T.The susceptibility detecyion technology of asthma disease in Xiang pigs was established on the basis of CD gene structural variation[D].Guiyang:Guizhou University,2024.(in Chinese)
[13] 朱丽飞.组织蛋白酶V溶酶体定位调控肺癌进展的作用和机制研究[D].广州：南方医科大学,2023.ZHU L F.Mechanism of cathepsin V lysomal localization contributions to the progression of lung cancer[D].Guangzhou:Southern Medical University,2023.(in Chinese)
[14] LENG Y P,MA Y S,LI X G,et al.L-homocysteine-induced cathepsin V mediates the vascular endothelial inflammation in hyperhomocysteinaemia[J].British Journal of Pharmacology,2018,175(8):1157-1172.
[15] KEEGAN P M,WILDER C L,PLATT M O.Tumor necrosis factor alpha stimulates cathepsin K and V activity via juxtacrine monocyte-endothelial cell signaling and JNK activation[J].Molecular and Cellular Biochemistry,2012,367(1-2):65-72.
[16] SEVENICH L,HAGEMANN S,STOECKLE C,et al.Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice[J].Biochimie,2010,92(11):1674-1680.
[17] XIA Y,GE M H,XIA L,et al.CTSV (cathepsin V) promotes bladder cancer progression by increasing NF-κB activity[J].Bioengineered,2022,13(4):10180-10190.
[18] YANG L W,ZENG Q,DENG Y,et al.Glycosylated cathepsin V serves as a prognostic marker in lung cancer[J].Frontiers in Oncology,2022,12:876245.
[19] SONG Z X,SHAH S,LV B X,et al.Anti-aging and anti-oxidant activities of murine short interspersed nuclear element antisense RNA[J].European Journal of Pharmacology,2021,912:174577.
[20] PARK E,MAQUAT L E.Staufen-mediated mRNA decay[J].Wiley Interdisciplinary Reviews-RNA,2013,4(4):423-435.
[21] LUCAS B A,LAVI E,SHIUE L,et al.Evidence for convergent evolution of SINE-directed Staufen-mediated mRNA decay[J].Proceedings of the National Academy of Sciences of the United States of America,2018,115(5):968-973.
[22] 周显珊,黄世会,牛熙,等.皱皮香猪泛素化连接酶2基因结构变异的差异表达研究[J].畜牧兽医学报,2025,56(1):136-146.ZHOU X S,HUANG S H,NIU X,et al.Differential expression study of structural varitions in the ubiquitin ligase 2 gene of Xiang pigs with wrinkled skin[J].Acta Veterinaria et Zootechnica Sinica,2025,56(1)：136-146.(in Chinese)
[23] LIU C,RAN X Q,NIU X,et al.Insertion of 275-bp SINE into first intron of PDIA4 gene is associated with litter size in Xiang pigs[J].Animal Reproduction Science,2018,195:16-23.
[24] 王金柱,赵一鸣,蒋琰,等.RNA结合蛋白在肿瘤中的作用[J].生命的化学,2024,44(9):1620-1628.WANG J Z,ZHAO Y M,JIANG Y,et al.Emerging roles of RNA-binding proteins in cancers[J].Chemistry of Life,2024,44(9):1620-1628.(in Chinese)
[25] ZHAO Y,MIR C,GARCIAMAYEA Y,et al.RNA-binding proteins:Underestimated contributors in tumorigenesis[J].Seminars in Cancer Biology,2022,86(Pt3):431-444.
[26] ZARNACK K,KÖNIG J,TAJNIK M,et al.Direct competition between hnRNP C and U2AF65 protects the transcriptome from the exonization of Alu elements[J].Cell,2013,152(3):453-466.
[27] MENESES A,KOGA S,O’LEARY J,et al.TDP-43 pathology in Alzheimer’s disease[J].Molecular Neurodegeneration,2021,16(1):84.
[28] BARTEL D P.microRNAs:Target recognition and regulatory functions[J].Cell,2009,136(2):215-233.
[29] MARTA C S,MONIKA G,DOBROCHNA D,et al.Deciphering miRNAs’ action through miRNA editing[J].International Journal of Molecular Sciences,2019,20(24):6249.
[30] FAKERI M,ARMANDZAD M R,OLYAEI S S,et al.The importance of miR-491-5p in various cancers[J].Current Molecular Medicine,2023,23(9):921-933.
[31] 李洪霞,刘芳,夏俊华.miR-670-5p对肺癌细胞增殖、迁移、侵袭的影响[J].中国应用生理学杂志,2021,37(5):500-505.LI H X,LIU F,XIA J H.Effect of miR-670-5p on proliferation,migration and invasion of lung cancer cells[J].Chinese Journal of Applied Physiology,2021,37(5):500-505.(in Chinese)
[32] SHI C L,XU X D.miR-670-5p induces cell proliferation in hepatocellular carcinoma by targeting PROX1[J].Biomedicine & Pharmacotherapy,2016,77:20-26.
[33] ZHOU L H,ZOU H,HAO J Y,et al.Metformin inhibits ovarian granular cell pyroptosis through the miR-670-3p/NOX2/ROS pathway[J].Aging,2023,15(10):4429-4443.
[34] 卫九健,郭玉新.猪呼吸系统疾病临床诊断与治疗[J/OL].畜牧兽医科学(电子版),2022,22:91-93.WEI J J,GOU Y X.Clinical diagnosis and treatment of swine respiratory diseases[J/OL].Graziery Veterinary Sciences (Electronic Version),2022,22:91-93.(in Chinese)