中国畜牧兽医 ›› 2025, Vol. 52 ›› Issue (7): 2981-2991.doi: 10.16431/j.cnki.1671-7236.2025.07.002

• 生物技术 • 上一篇    下一篇

gga-miR-1574-5p生物学功能预测及其与G3BP2基因靶向关系验证

平玉宇1,2, 黄烜1, 蔡清清1, 王强州1, 王佳兴2, 白皓1, 陈世豪1, 常国斌1   

  1. 1. 扬州大学动物科学与技术学院, 扬州 225009;
    2. 扬州大学兽医学院, 扬州 225009
  • 收稿日期:2024-09-26 出版日期:2025-07-05 发布日期:2025-07-01
  • 通讯作者: 陈世豪 E-mail:mrrchen@yzu.edu.cn
  • 作者简介:平玉宇,E-mail:32382011988@qq.com。
  • 基金资助:
    国家重点研发计划(2022YFD1800303);国家肉鸡产业技术体系(CARS-41)

Prediction of Biological Function of gga-miR-1574-5p and Verification of Its Targeting Relationship with G3BP2 Gene

PING Yuyu1,2, HUANG Xuan1, CAI Qingqing1, WANG Qiangzhou1, WANG Jiaxing2, BAI Hao1, CHEN Shihao1, CHANG Guobin1   

  1. 1. College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China;
    2. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China
  • Received:2024-09-26 Online:2025-07-05 Published:2025-07-01

摘要: 【目的】 gga-miR-1574-5p是细胞营养匮乏反应相关的miRNA。本研究旨在分析gga-miR-1574-5p功能并鉴定其与G3BP2基因之间的靶向关系,为进一步解析gga-miR-1574-5p的生物学功能提供科学依据。【方法】 通过在线工具miRBase获得gga-miR-1574-5p的成熟序列;使用TargetScan数据库预测gga-miR-1574-5p的靶基因进行GO功能与KEGG通路富集分析,并预测其与G3BP2基因的结合位点;使用双荧光素酶报告基因试验验证gga-miR-1574-5p与G3BP2的靶向关系;运用实时荧光定量PCR检测过表达gga-miR-1574-5p对G3BP2基因表达影响;采用Western blotting检测鸡巨噬细胞中gga-miR-1574-5p对G3BP2蛋白的表达影响。【结果】 GO功能富集显示,gga-miR-1574-5p的潜在靶基因主要富集于细胞核、细胞质膜等细胞组分;RNA聚合酶Ⅱ的转录调控、RNA聚合酶Ⅱ的正向转录调控等生物过程;以及蛋白质结合、ATP结合等分子功能。KEGG通路富集显示,靶基因主要富集到卵母细胞减数分裂、TGF-β信号通路和线粒体自噬通路等。TargetScan数据库检测到gga-miR-1574-5p种子区与鸡G3BP2基因3'-非翻译区(3'-UTR)存在结合位点;与G3BP2-3'-UTR野生型质粒和NC-mimics共转染组相比,G3BP2-3'-UTR野生型质粒和gga-miR-1574-5p mimics共转染组的双荧光素酶活性极显著降低(P<0.01)。实时荧光定量PCR结果表明,与NC-mimics组相比,转染20 nmol/L gga-miR-1574-5p mimics后对G3BP2基因表达水平无显著影响(P>0.05),而转染30、50 nmol/L gga-miR-1574-5p mimics组中G3BP2基因表达量显著或极显著降低(P<0.05;P<0.01)。Western blotting结果显示,转染gga-miR-1574-5p mimics(20、30、50 nmol/L)后G3BP2蛋白表达水平均极显著低于转染NC-mimics组(P<0.01)。【结论】 gga-miR-1574-5p与G3BP2基因存在靶向关系,且gga-miR-1574-5p能通过与G3BP2-3'-UTR特异性结合抑制鸡巨噬细胞中G3BP2基因及蛋白的表达。

关键词: gga-miR-1574-5p; 靶基因; G3BP2基因; 应激颗粒

Abstract: 【Objective】 gga-miR-1574-5p is a miRNA associated with the cellular nutrient deprivation response.This study was aimed to analyze the function of gga-miR-1574-5p and identify its targeting relationship with G3BP2 gene,so as to provide a scientific basis for further analysis of the biological function of gga-miR-1574-5p. 【Method】 The mature sequence of gga-miR-1574-5p was obtained by online tool miRBase.Target genes of gga-miR-1574-5p were predicted using TargetScan database for GO function and KEGG pathway enrichment analysis,and the binding sites of G3BP2 gene and gga-miR-1574-5p were predicted.Dual luciferase reporter gene assay was used to verify the targeting relationship between gga-miR-1574-5p and G3BP2.Real-time quantitative PCR was used to detect the effect of gga-miR-1574-5p overexpression on G3BP2 gene expression.The effect of gga-miR-1574-5p on the expression of G3BP2 protein in chicken macrophages was detected by Western blotting. 【Result】 GO function enrichment results showed that the potential target genes of gga-miR-1574-5p were mainly enriched in cellular components such as nucleus and plasma membrane,biological processes such as regulation of transcription by RNA polymerase Ⅱ and positive regulation of transcription by RNA polymerase Ⅱ,and molecular functions such as protein binding and ATP binding.KEGG pathway enrichment results showed that the target genes were mainly concentrated in oocyte meiosis,TGF-beta signaling pathway,mitophagy-animal pathway,etc.A binding site between gga-miR-1574-5p seed region and the 3'-untranslated region (3'-UTR) of G3BP2 gene in Gallus gallus was detected in TargetScan database.Compared with G3BP2-3'-UTR wild-type plasmid and NC-mimics co-transfection group,the dual luciferase activity of G3BP2-3'-UTR wild-type plasmid and gga-miR-1574-5p mimics co-transfection group was extremely significantly decreased (P<0.01).Real-time quantitative PCR results showed that compared with NC-mimics group,transfected with 20 nmol/L gga-miR-1574-5p mimics had no significant effect on the expression of G3BP2 gene (P>0.05),but the expression of G3BP2 gene in 30 and 50 nmol/L gga-miR-1574-5p mimics groups were significantly or extremely significantly decreased (P<0.05 or P<0.01).Western blotting results showed that the expression of G3BP2 protein in 20,30 and 50 nmol/L gga-miR-1574-5p mimics groups were extremely significantly lower than that in NC-mimics group (P<0.01). 【Conclusion】 gga-miR-1574-5p had a targeting relationship with G3BP2 gene,and gga-miR-1574-5p could inhibit the expression of G3BP2 gene and protein in chicken macrophages by specifically binding to G3BP2-3'-UTR.

Key words: gga-miR-1574-5p; target gene; G3BP2 gene; stress granules

中图分类号: