miR-144-5p在不同体型双峰驼血浆外泌体中的表达及靶基因验证

doi:10.16431/j.cnki.1671-7236.2025.03.005

摘要/Abstract

摘要： 【目的】探讨双峰驼血浆外泌体中miR-144-5p在脂质代谢中的潜在调控机制。【方法】采用超速离心法分离不同肥瘦程度的两组双峰驼血浆外泌体后，运用透射电子显微镜和纳米颗粒跟踪分析技术对分离的外泌体进行形态学观察与鉴定。提取外泌体总RNA后，进行高通量测序，鉴定筛选出在两组双峰驼血浆外泌体中差异表达的miRNA。应用TargetScan和miRWalk在线软件预测差异表达miRNA的靶基因，并利用DAVID和KEGG在线分析系统对靶基因进行GO功能和KEGG通路富集分析。构建含靶位点的野生型载体和突变型表达质粒载体，通过miR-144-5p与293T细胞共转染进行双荧光素酶相对活性检测，验证miR-144-5p与其候选基因的作用关系，探讨其在脂质代谢调控中的作用机制。【结果】双峰驼血浆外泌体呈杯状或圆形，直径约100 nm，粒径分布范围为30～150 nm；两组双峰驼外泌体间共有40个差异表达miRNAs，miR-144-5p是差异最显著的miRNA (P＜0.01)；miR-144-5p序列在不同物种间高度保守；预测其靶基因是过氧化物酶体增殖受体γ辅激活因子α(PGC-1α)，该基因主要参与胆固醇平衡和脂质磷酸化等过程，富集于甘油酯代谢、胆固醇代谢和动脉粥样硬化等信号通路；miR-144-5p对野生型PGC-1α-3'-UTR载体的荧光素酶相对活性具有极显著抑制作用(P＜0.01)，而在突变型PGC-1α重组质粒组中，转染后48 h时，miR-144-5p的荧光素酶活性与阴性对照组相比无显著差异(P＞0.05)，表明miR-144-5p通过结合PGC-1α基因种子序列来抑制PGC-1α的表达。【结论】本研究揭示了miR-144-5p在不同体型双峰驼血浆外泌体中的表达差异，并证实其通过调控PGC-1α在脂质代谢中发挥重要作用。

关键词: 双峰驼; 外泌体; miR-144-5p; 脂质代谢; 靶基因; 双荧光素酶报告系统

Abstract: 【Objective】 The aim of this study was to explore the potential regulatory mechanisms of miR-144-5p in lipid metabolism within plasma exosomes of Bactrian camels.【Method】 The exosomes were separated from two groups of Bactrian camels with different levels of fat and lean using ultracentrifugation in this study.The morphology of the exosomes was observed by transmission electron microscopy and identified using nanoparticle tracking analysis.Total exosomal RNA was extracted for small RNA sequencing,and the differentially expressed miRNA in plasma exosomes from two groups of Bactrian camels was selected.TargetScan and miRWalk online software were used to predict candidate target gene of differentially expressed miRNA,followed by GO function and KEGG pathway enrichment analysis of the target genes using DAVID and KEGG bioinformatics systems.The wild-type vectors containing target sites and mutant expression plasmids were constructed.The transfection of miR-144-5p minicis with 293T cells was done for dual-luciferase reporter assay to validate the relationship between miR-144-5p and its candidate gene,exploring its mechanism of action in the regulation of lipid metabolism.【Result】 The results revealed that plasma exosomes from Bactrian camels exhibited cup-shaped or round morphology with a diameter of approximately 100 nm,with a particle size distribution ranging between 30 to 150 nm.A total of 40 differentially expressed miRNAs were identified between the two groups,including significant differential expression of miR-144-5p (P＜0.01).The sequence of miR-144-5p was highly conserved among different species.PGC-1α was predicted as its target gene,mainly involved in processes like cholesterol balance and lipid phosphorylation,enriched in glycerolipid metabolism,cholesterol metabolism,and atherosclerosis signaling pathways.miR-144-5p significantly inhibited the relative luciferase activity of the wild-type PGC-1α-3'-UTR vector (P＜0.01),while it had no effect on the mutant PGC-1α-3'-UTR vector compared to the negative control group at 48 hours after transfection (P＞0.05),indicating that miR-144-5p inhibits PGC-1α gene expression by binding to the gene’s seed sequence.【Conclusion】 This study revealed the expression differences of miR-144-5p in plasma exosomes of Bactrian camels with different body types and confirmed its important role in lipid metabolism by regulating PGC-1α.

Key words: Bactrian camels; exosome; miR-144-5p; lipid metabolism; target gene; dual-luciferase reporter assay system

中图分类号:

S852.2

斯日古楞, 于雯, 降晓薇, 李子怡, 金君健, 白浩宇. miR-144-5p在不同体型双峰驼血浆外泌体中的表达及靶基因验证[J]. 中国畜牧兽医, 2025, 52(3): 1022-1032.

Siriguleng, YU Wen, JIANG Xiaowei, LI Ziyi, JIN Junjian, BAI Haoyu. The Expression of miR-144-5p in Plasma Exosomes of Different Body Types of Bactrian Camels and the Verification of Target Gene[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(3): 1022-1032.

参考文献

[1] MATHIEU M,MARTIN-JAULAR L,LAVIEU G,et al.Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication[J].Nature Cell Biology,2019,21(1):9-17.
[2] MATSUMURA T，SUGIMACHI K，IINUMA H, et al. Exosomal microRNA in serum is a novel biomarker of recurrence in human colorectal cancer[J].British Journal of Cancer,2015,113(2):275-281.
[3] YAO H,YU S,LUO Y,et al.Effects of plasma-derived exosomes from the normal and thin Bactrian camels on hepatocellular carcinoma and their differences at transcriptome and proteomics levels[J].Frontiers in Oncology,2023,13:994340.
[4] WAGENER M G,GANTER M,LEONHARD-MAREK S.Body condition scoring in alpacas (Vicugna pacos) and llamas (Lama glama)—A scoping review[J].Veterinary Research Communications,2024,48(2):665-684.
[5] PADALINO B,MENCHETTI L.The first protocol for assessing welfare of camels[J].Frontiers in Veterinary Science,2020,7:631876.
[6] ALI M A,ABU D H,ADEM M A,et al. Effects of long-term dehydration on stress markers,blood parameters,and tissue morphology in the dromedary camel (Camelus dromedarius)[J].Frontiers in Veterinary Science,2023,10:1236425.
[7] ZHANG B,YANG Y,XIANG L,et al.Adipose-derived exosomes:A novel adipokine in obesity-associated diabetes[J].Journal of Cellular Physiology,2019,234(10):16692-16702.
[8] BADAWY A A,EL-MAGD M A,ALSADRAH S A.Therapeutic effect of camel milk and its exosomes on MCF7 cells in vitro and in vivo[J].Integrative Cancer Therapies,2018,17(4):1235-1246.
[9] EL-KATTAWY A M,ALGEZAWY O,ALFAIFI M Y, et al.Therapeutic potential of camel milk exosomes against HepaRG cells with potent apoptotic,anti-inflammatory,and anti-angiogenesis effects for colostrum exosomes[J].Biomedicine & Pharmacotherapy，2021,143:112220.
[10] KALLURI R,LEBLEU V S.The biology,function,and biomedical applications of exosomes[J].Science,2020,367(6478):eaau6977.
[11] ORTEGA F J,MERCADER J M,CATALAN V, et al. Targeting the circulating microRNA signature of obesity[J].Clinical Chemistry,2013,59(5):781-792.
[12] CHOI W H,AHN J,UM M Y,et al.Circulating microRNA expression profiling in young obese Korean women[J].Nutrition Research and Practice,2020,14(4):412-422.
[13] ASSMANN T S,RIEZU-BOJ J I,MILAGRO F I,et al. Circulating adiposity-related microRNAs as predictors of the response to a low-fat diet in subjects with obesity[J].Journal of Cellular and Molecular Medicine,2020,24(5):2956-2967.
[14] CHRISTODOULOU F,RAIBLE F,TOMER R, et al.Ancient animal microRNAs and the evolution of tissue identity[J].Nature,2010,463(7284):1084-1088.
[15] NI HY,YU L,ZHAO X L, et al.Seed oil of Rosa roxburghii Tratt against non-alcoholic fatty liver disease in vivo and in vitro through PPARα/PGC-1α-mediated mitochondrial oxidative metabolism[J].Phytomedicine,2022,98:153919.
[16] WU H,LIU B,CHEN Z,et al. MSC-induced lncRNA HCP5 drove fatty acid oxidation through miR-3619-5p/AMPK/PGC1α/CEBPB axis to promote stemness and chemo-resistance of gastric cancer[J].Cell Death and Disease，2020,11(4):233.