猪miR-1343生物信息学分析及其在卵泡颗粒细胞中的表达研究

doi:10.16431/j.cnki.1671-7236.2025.08.022

摘要/Abstract

摘要： 【目的】为探究miR-1343在猪卵巢颗粒细胞中的调控机制，本研究基于生物信息学方法对miR-1343靶基因进行预测分析，并探索生殖激素对猪卵巢颗粒细胞中miR-1343表达的影响。【方法】通过miRbase在线数据库获取miR-1343成熟序列，并进行序列保守性分析。分离培养猪卵泡颗粒细胞，接种于6孔板，待细胞汇合度达70%～80%时，将细胞分为2组：对照组和试验组，分别对应添加0和20 IU/mL卵泡刺激素(follicle-stimulating hormone,FSH)，每组3个重复，添加24 h后收集细胞，利用实时荧光定量PCR检测miR-1343的表达水平。利用PROMO在线软件对猪miR-1343启动子区转录因子结合位点进行预测。通过TargetScan、miRDB和Starbase数据库对miR-1343进行靶基因预测，并利用DAVID在线数据库对靶基因进行GO功能和KEGG通路富集分析。采用STRING数据库对靶基因进行蛋白互作分析(PPI)，采用Cytoscape软件绘制蛋白质调控网络。【结果】 miR-1343成熟序列在多个物种中高度保守。与对照组相比，试验组细胞miR-1343的表达极显著下调(P＜0.01)。miR-1343启动子区分布有转录因子Sp1、SMAD3、SMAD4和FOXO4等结合位点。选用不同数据库预测共得到278个靶基因。GO功能富集结果显示，miR-1343靶基因主要富集在磷酸化、RNA聚合酶Ⅱ启动子转录的正调控、蛋白质磷酸化、DNA模板转录的正调控等生物过程；细胞核、细胞质、细胞质基质、核质等细胞组分；以及ATP结合、蛋白质同源二聚活性、蛋白丝氨酸/苏氨酸激酶活性、染色质结合等分子功能。KEGG通路富集结果显示，miR-1343靶基因显著富集到与卵巢功能高度相关的多条信号通路，如Hippo信号通路、MAPK信号通路、甲状腺激素信号通路等。miR-1343靶基因PPI分析结果显示，SYNJ1、PLCB1、GSK3B基因与其他蛋白存在较多靶向关系。【结论】颗粒细胞中miR-1343的表达受FSH的影响。miR-1343靶基因显著富集于卵巢功能相关的多条信号通路，miR-1343可能通过调控靶基因PLCB1、GSK3B的表达进而参与猪卵泡颗粒细胞的发育。本研究结果为进一探究miR-1343在猪卵巢颗粒细胞中的调控机制提供参考依据。

关键词: 猪卵泡颗粒细胞; miR-1343; 靶基因; 生物信息学

Abstract: 【Objective】 The purpose of this paper was to investigate the regulatory mechanism of miR-1343 in porcine ovarian granulosa cells.The target genes of miR-1343 were predicted and analyzed by the bioinformatics method，and the effects of reproductive hormone on miR-1343 expression were explored in porcine ovarian granulosa cells. 【Method】 Mature sequences of miR-1343 were downloaded from the miRbase online database for sequence conservative analysis.Porcine ovarian granulosa cells were isolated and cultured,which were seeded into six-well plates.At 70%-80% cell confluence,the granulosa cells were treated with 0 IU/mL (control group) and 20 IU/mL (experimental group) follicle-stimulating hormone (FSH),with 3 replicates per group,24 h after addition,the cells were harvested,and the expression of miR-1343 was detected by Real-time quantitative PCR.The PROMO online software was utilized to predict the transcription factor binding sites for miR-1343 in pigs.The target genes of miR-1343 were predicted using TargetScan,miRDB,and Starbase database.GO function and KEGG pathway enrichment analysis were performed for the target genes using DAVID online database.The protein-protein interaction (PPI) of target genes was analyzed using STRING database,and regulatory network was constructed using Cytoscape software. 【Result】 The mature sequences of miR-1343 were highly conserved among multiple species.The relative expression of miR-1343 in experimental group was extremely significantly down-regulated compared with the control group (P＜0.01).There were many transcription factor binding sites such as Sp1,SMAD3,SMAD4,and FOXO4 in the promoter region of miR-1343.A total of 278 common target genes were predicted using different databases.GO function annotation analysis results revealed that target genes of miR-1343 were mainly enriched in phosphorylation,positive regulation of transcription by RNA polymerase Ⅱ,protein phosphorylation,and positive regulation of DNA-templated transcription for biological process,nucleus,cytoplasm,cytosol,and nucleoplasm for cellular component,and ATP binding,protein homodimerization activity,protein serine/threonine kinase activity and chromatin binding for molecular function.KEGG analysis results revealed that target genes of miR-1343 were significantly enriched in the Hippo signaling pathway,MAPK signaling pathway,and thyroid hormone signaling pathway,which were related to ovarian function.PPI analysis of miR-1343 target genes indicated that SYNJ1,PLCB1,and GSK3B had more targeted relationships with other proteins. 【Conclusion】 The expression of miR-1343 was influenced by FSH stimulation in granulosa cells.miR-1343 might participate in the development of porcine ovarian granulosa cells by regulating the expression of target genes such as PLCB1 and GSK3B.The results provided a reference for further exploring the regulatory mechanism of miR-1343 in porcine ovarian granulosa cells.

Key words: porcine ovarian granulosa cells; miR-1343; target gene; bioinformatics

中图分类号:

S813.3

胡慧艳, 邵丽玮, 刘小辉, 袁英, 邳明伟. 猪miR-1343生物信息学分析及其在卵泡颗粒细胞中的表达研究[J]. 中国畜牧兽医, 2025, 52(8): 3734-3743.

HU Huiyan, SHAO Liwei, LIU Xiaohui, YUAN Ying, PI Mingwei. Bioinformatics Analysis of miR-1343 and Its Expression in Porcine Ovarian Granulosa Cells[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(8): 3734-3743.

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