秦川肉牛PAI1基因生物信息学分析及其对肌内脂肪细胞增殖和分化的影响

doi:10.16431/j.cnki.1671-7236.2023.05.001

摘要/Abstract

摘要： 【目的】对秦川肉牛纤溶酶原激活物抑制剂1(plasminogen activator inhibitor 1,PAI1)基因进行生物信息学分析,检测PAI1基因在秦川肉牛不同组织中的表达水平并研究其功能,为秦川肉牛肌内脂肪沉积的分子育种提供理论依据。【方法】以分离自秦川肉牛背最长肌的肌内脂肪细胞为试验材料,PCR扩增PAI1基因CDS区,对所获序列进行相似性分析及系统进化树构建,并通过生物信息学软件预测其编码蛋白的结构和功能。通过实时荧光定量PCR检测PAI1基因在秦川肉牛不同组织中的表达量。合成PAI1基因siRNA并转染秦川肉牛肌内脂肪细胞,试验分为两组:对照组(NC)和干扰组(si-PAI1),通过EdU染色、CCK-8检测、流式细胞术、油红O染色观察表型变化,采用实时荧光定量PCR及Western blotting检测转录和翻译水平标志基因(PCNA、CDK1、CCND2、PPARG、PLIN2、FABP4)的表达。【结果】试验成功扩增出PAI1基因CDS区,大小为1 054 bp。牛PAI1基因氨基酸序列与水牛、山羊、人、小鼠、绵羊、黑猩猩的相似性分别为96.8%、95.8%、85.3%、76.9%、95.8%和85.3%。系统进化树表明,牛与水牛的亲缘关系最近,与小鼠的亲缘关系最远。生物信息学分析发现,细胞周期相关激酶参与了PAI1基因的磷酸化修饰;PAI1蛋白二级结构主要以α-螺旋和无规则卷曲为主;PAI1基因启动子上游2 000 bp存在成脂相关转录因子的结合位点。实时荧光定量PCR结果发现,PAI1基因在秦川肉牛各组织中均有表达,其中在肾周脂肪中的表达量最高,在背最长肌中的表达量最低。干扰PAI1基因后,与对照组相比,干扰组处于增殖期的细胞数量明显增多,CDK1、PCNA和CCND2基因mRNA表达水平极显著或显著上调(P＜0.01;P＜0.05),CDK1和PCNA蛋白表达水平明显增加。随着肌内脂肪细胞的分化,与对照组相比,干扰组脂滴数量明显增加,PPARγ、PLIN2和FABP4基因mRNA表达水平均极显著上调(P＜0.01)。【结论】PAI1基因在物种间的保守性较强,可能通过磷酸化和转录因子参与肌内脂肪细胞的增殖和分化,干扰PAI1基因可促进肌内脂肪细胞的增殖和分化,提示该基因可能对肌内脂肪的沉积有重要的调控作用。

关键词: 秦川肉牛; PAI1基因; 生物信息学; 肌内脂肪细胞

Abstract: 【Objective】 The purpose of this experiment was to conduct bioinformatics analysis of plasminogen activator inhibitor 1 (PAI1) gene in Qinchuan beef cattle,detect its expression in different tissues and confirm its function by experiments,which provided the theoretical basis for molecular breeding to promote intramuscular fat deposition in Qinchuan beef cattle.【Method】 Intramuscular adipocytes were isolated from longissimus dorsi muscle of Qinchuan beef cattle as the research object.The CDS region of PAI1 gene was amplified by PCR.The similarity was analyzed and the phylogenetic tree was constructed,the structure and function of the encoding protein of PAI1 gene was predicted by bioinformatics software.The expression of PAI1 gene in different tissues of Qinchuan beef cattle was detected by Real-time quantitative PCR.The intramuscular adipocytes was transfected with siRNA of PAI1 gene,the experiment was divided into two groups:Control group (NC) and interference group (si-PAI1).Flow cytometry,EdU staining,CCK-8 assay,and Oil red O staining were used to detect the cell phenotypic alterations.Real-time quantitative PCR and Western blotting were used to identify the expression of marker genes (PCNA,CDK1,CCND2,PPARG,PLIN2 and FABP4) at the transcriptional and translational levels.【Result】 The CDS region (1 054 bp) of PAI1 gene was successfully amplified.The similarity of amino acid sequence of PAI1 protein between Bos taurus and Bubalus bubalis,Capra hircus,Homo sapiens,Mus musculus,Ovis aries and Pan troglodytes were 96.8%,95.8%,85.3%,76.9%,95.8% and 85.3%,respectively.The phylogenetic tree results showed that Bos taurus had the closest genetic relationship with Bubalus bubalis,and the farthest genetic relationship with Mus musculus.Bioinformatics prediction showed that cell cycle-related kinase was involved in the phosphorylation of PAI1 gene.The secondary structure of PAI1 protein were mainly alpha helix and random coil.The binding sites of adipogenic transcription factors were also identified in the upstream 2 000 bp of PAI1 gene promoter.Real-time quantitative PCR results showed that PAI1 gene was expressed in all tissues,with the highest expression in perirenal fat and the lowest expression in longissimus dorsi muscle.After interfering with PAI1 gene,compared with control group,the cell number of the proliferative phase in interference group was significantly increased,the mRNA expression of CDK1,PCNA and CCND2 genes were extremely significantly or significantly increased (P＜0.01 or P＜0.05),and the protein expression of CDK1 and PCNA were also significantly increased.With the differentiation of intramuscular adipocytes,compared with control group,the number of lipid droplets in interference group was significantly increased,and the mRNA expression of PPARγ,PLIN2 and FABP4 genes were extremely significantly increased (P＜0.01).【Conclusion】 PAI1 gene was highly conserved among species.PAI1 might participate in the proliferation and differentiation of intramuscular adipocytes through phosphorylation and transcription factors.The interfering with PAI1 gene could promote the proliferation and differentiation of intramuscular adipocytes,which suggested that PAI1 gene might play an important role in regulating the deposition of intramuscular fat.

Key words: Qinchuan beef cattle; PAI1 gene; bioinformatics; intramuscular adipocytes

中图分类号:

张殿琦, 马鑫浩, 杨志梅, 梅楚刚, 昝林森. 秦川肉牛PAI1基因生物信息学分析及其对肌内脂肪细胞增殖和分化的影响[J]. 中国畜牧兽医, 2023, 50(5): 1729-1741.

ZHANG Dianqi, MA Xinhao, YANG Zhimei, MEI Chugang, ZAN Linsen. Bioinformatics Analysis of PAI1 Gene and Its Effect on the Proliferation and Differentiation of Intramuscular Adipocytes in Qinchuan Beef Cattle[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(5): 1729-1741.

参考文献

[1] 昝林森,梅楚刚,王洪程.我国肉牛产业经济面临的形势及对策建议[A].第十八届中国科协年会——分14西北地区农牧结合发展草牧业研讨会论文集[C].2016. ZAN L S,MEI C G,WANG H C.Economic situation,existing problems and countermeasures on development of beef cattle industry in China[A].The 18th China Association for Science and Technology Annual Conference-Part 14 Discussion on the Development of Grass Animal Husbandry in Northwest China Proceedings of the Conference[C].2016.(in Chinese)
[2] 池永宽,刘旭光,熊康宁,等.影响肉牛胴体品质候选基因研究进展[J].家畜生态学报,2022,43(5):6-13. CHI Y K,LIU X G,XIONG K N,et al.Research progress on candidate genes affecting carcass quality of beef cattle[J].Acta Ecologae Animalis Domastici,2022,43(5):6-13.(in Chinese)
[3] FRANK D,KACZMARSKA K,PATERSON J,et al.Effect of marbling on volatile generation,oral breakdown and in mouth flavor release of grilled beef[J].Meat Science,2017,133:61-68.
[4] SAN J,DU Y,WU G,et al.Transcriptome analysis identifies signaling pathways related to meat quality in broiler chickens-the extracellular matrix (ECM) receptor interaction signaling pathway[J].Journal of Poultry Science,2021,100(6):101135.
[5] ORELLANA C,PEÑA F,GARCÍA A,et al.Carcass characteristics,fatty acid composition,and meat quality of Criollo Argentino and Braford steers raised on forage in a semi-tropical region of Argentina[J].Meat Science,2009,81(1):57-64.
[6] INDURAIN G,BERIAIN M J,GOÑI M V,et al.Composition and estimation of intramuscular and subcutaneous fatty acid composition in Spanish young bulls[J].Meat Science,2006,73(2):326-334.
[7] KONG H J,KWON E J,KWON O S,et al.Crosstalk between YAP and TGFβ regulates SERPINE1 expression in mesenchymal lung cancer cells[J].International Journal of Oncology,2021,58(1):111-121.
[8] LARA-CASTRO C,FU Y,CHUNG B H,et al.Adiponectin and the metabolic syndrome:Mechanisms mediating risk for metabolic and cardiovascular disease[J].Current Opinion in Lipidology,2007,18(3):263-270.
[9] ALESSI M C,POGGI M,JUHAN-VAGUE I.Plasminogen activator inhibitor-1,adipose tissue and insulin resistance[J]. Current Opinion in Lipidology,2007,18(3):240-245.
[10] GUGLIUCCI A.Biomarkers of dysfunctional visceral fat[J].Advances in Clinical Chemistry,2022,109:1-30.
[11] TENG F,ZHANG J X,CHEN Y,et al.lncRNA NKX2-1-AS1 promotes tumor progression and angiogenesis via upregulation of SERPINE1 expression and activation of the VEGFR-2 signaling pathway in gastric cancer[J].Molecular Oncology,2021,15(4):1234-1255.
[12] XU Y,CHEN W,LIANG J,et al.The miR-1185-2-3p-GOLPH3L pathway promotes glucose metabolism in breast cancer by stabilizing p53-induced SERPINE1[J].Journal of Experimental&Clinical Cancer Research,2021,40(1):47.
[13] MCCANN J V,XIAO L,KIM D J,et al.Endothelial miR-30c suppresses tumor growth via inhibition of TGF-β-induced Serpine1[J].Journal of Clinical Investigation,2019,129(4):1654-1670.
[14] LIANG X,KANJANABUCH T,MAO S L,et al.Plasminogen activator inhibitor-1 modulates adipocyte differentiation[J]. American Journal of Physiology-Endocrinology and Metabolism,2006,290(1):E103-E113.
[15] KARBIENER M,NEUHOLD C,OPRIESSNIG P,et al.microRNA-30c promotes human adipocyte differentiation and co-represses PAI-1 and ALK2[J].RNA Biology,2011,8(5):850-860.
[16] HUANG W,GUO Y,DU W,et al.Global transcriptome analysis identifies differentially expressed genes related to lipid metabolism in Wagyu and Holstein cattle[J].Scientific Reports,2017,7(1):5278.
[17] DU Y,WANG Y,XU Q,et al.TMT-based quantitative proteomics analysis reveals the key proteins related with the differentiation process of goat intramuscular adipocytes[J].BMC Genomics,2021,22(1):417.
[18] WANG X,LIANG C,LI A,et al.RNA-Seq and lipidomics reveal different adipogenic processes between bovine perirenal and intramuscular adipocytes[J].Adipocyte,2022,11(1):448-462.
[19] 鄢婉容.PAI-1基因对C2C12成肌细胞增殖、凋亡和分化的影响[D].长沙:湖南农业大学,2020. YAN W R.Effects of PAI-1 gene on the proliferation,apoptosis and differentiation of C2C12 myoblasts[D].Changsha:Hunan Agricultural University,2020.(in Chinese)
[20] 赵丽芳,陶美林,潘国庆.丝氨酸蛋白酶抑制剂超家族的研究进展[J].蚕业科学,2016,42(3):532-540. ZHAO L F,TAO M L,PAN G Q.Research progress of serine protease inhibitor superfamily[J].Sericulture Science,2016,42(3):532-540.(in Chinese)
[21] BARNARD S A,PIETERS M,DE LANGE Z.The contribution of different adipose tissue depots to plasma plasminogen activator inhibitor-1(PAI-1) levels[J].Blood Reviews,2016,30(6):421-429.
[22] IBRAHIM M M.Subcutaneous and visceral adipose tissue:Structural and functional differences[J].Obesity Reviews,2010,11(1):11-18.
[23] KOURI F M,QUEISSER M A,KÖNIGSHOFF M,et al.Plasminogen activator inhibitor type 1 inhibits smooth muscle cell proliferation in pulmonary arterial hypertension[J].International Journal of Biochemistry and Cell Biology,2008,40(9):1872-1882.
[24] PLOPLIS V A,BALSARA R,SANDOVAL-COOPER M J,et al.Enhanced in vitro proliferation of aortic endothelial cells from plasminogen activator inhibitor-1-deficient mice[J].Journal of Biological Chemistry,2004,279(7):6143-6151.
[25] ZHANG H,WU G,FENG J,et al.Expression of STOML2 promotes proliferation and glycolysis of multiple myeloma cells via upregulating PAI-1[J]. Journal of Orthopaedic Surgery and Research,2021,16(1):667.
[26] WANG L,SHAN T.Factors inducing transdifferentiation of myoblasts into adipocytes[J].Journal of Cellular Physiology,2021,236(4):2276-2289.
[27] LIJNEN H R,ALESSI M C,VAN HOEF B,et al.On the role of plasminogen activator inhibitor-1 in adipose tissue development and insulin resistance in mice[J].Journal of Thrombosis and Haemostasis,2005,3(6):1174-1179.
[28] ZOU M L,TENG Y Y,CHEN Z H,et al.The uPA system differentially alters fibroblast fate and profibrotic ability in skin fibrosis[J].Frontiers in Immunology,2022,13:845956.
[29] HADADEH O,BARRUET E,PEIRETTI F,et al.The plasminogen activation system modulates differently adipogenesis and myogenesis of embryonic stem cells[J].PLoS One,2012,7(11):e49065.