脂多糖与钙离子共处理的牛乳腺上皮细胞miRNA表达谱的比较分析

doi:10.16431/j.cnki.1671-7236.2024.09.035

摘要/Abstract

摘要： 【目的】探索钙离子对牛乳腺上皮细胞炎症反应的调控作用，比较分析脂多糖与钙离子共处理的牛乳腺上皮细胞的miRNA表达谱。【方法】使用脂多糖处理牛乳腺上皮细胞构建炎症反应模型，检测不同浓度(0.5～6.6 mmol/L)钙离子和脂多糖(50 μg/mL)共处理的牛乳腺上皮细胞的炎症因子转录水平。提取正常、脂多糖处理、脂多糖与钙离子共处理牛乳腺上皮细胞样本中总RNA，每组各3个样本。对9个RNA样本进行质量评估，并对其开展miRNA测序和生物信息学分析，最后对3组共有差异表达miRNA进行实时荧光定量PCR验证。【结果】在牛乳腺上皮细胞炎症模型中，高浓度钙离子可抑制炎症因子的转录。9个样本的clean reads在raw reads中占比为93.86%～98.18%，序列分布长度均集中在21～24 nt之间。3组样本的组内相似性高，而组间差异度高。3组样本共鉴定出47个差异表达miRNA，共有差异表达miRNA分别为bta-miR-11980、bta-miR-1246和bta-miR-677。GO功能和KEGG通路富集分析显示，差异表达miRNA的靶基因显著富集于细胞通讯调节、信号调节、内膜系统、系统发育、细胞发育过程、细胞分化等GO条目，显著富集于HIF-1信号通路、ErbB信号通路、TNF信号通路、突触囊泡周期、肌醇磷酸代谢途径等通路。实时荧光定量PCR验证结果显示，3组样本共有差异表达miRNA(bta-miR-677和bta-miR-1246)表达趋势与转录组测序结果一致。【结论】钙离子通过调控牛乳腺上皮细胞内bta-miR-11980、bta-miR-1246和bta-miR-677等差异表达miRNA的表达模式，间接影响牛乳腺上皮细胞炎症反应进程。

关键词: 脂多糖; 钙离子; 牛乳腺上皮细胞; miRNA表达谱

Abstract: 【Objective】 This study was aimed to investigate the regulation of calcium ions on inflammatory responses in bovine mammary epithelial cells, and compare and analyze miRNA expression profiles of bovine mammary epithelial cells co-treated with lipopolysaccharide and calcium ions. 【Method】 A model of inflammatory response was created by treating bovine mammary epithelial cells with lipopolysaccharide.The transcript levels of inflammatory factors were measured in bovine mammary epithelial cells co-treated with different concentrations (0.5-6.6 mmol/L) of calcium ions and lipopolysaccharide (50 μg/mL).RNA was extracted from bovine mammary epithelial cell samples with normal, lipopolysaccharide treated and co-treated with lipopolysaccharide and calcium ions, with 3 samples in each group.Quality assessment was performed on 9 RNA samples, followed by miRNA sequencing and bioinformatics analysis.Finally, Real-time quantitative PCR validation was performed on common differentially expressed miRNA among 3 groups. 【Result】 In a model of inflammation in bovine mammary epithelial cells, high concentrations of calcium ions were found to inhibit the transcription of inflammatory factors.Nine RNA samples were obtained, with clean reads accounting for between 93.86% and 98.18% of raw reads.The sequence distribution lengths were all concentrated between 21 and 24 nt.The samples from all three groups exhibited high intra-group similarity but also high inter-group variability.A total of 47 miRNA were identified as differentially expressed across 3 groups of samples.The samples among 3 groups showed differential expression of bta-miR-11980, bta-miR-1246 and bta-miR-677.The results of GO function and KEGG pathway enrichment analysis showed that the target genes of differentially expressed miRNA were significantly enriched in GO entries related to regulation of cell communication, signaling regulation, endomembrane system, phylogeny, cellular developmental process and cell differentiation, etc.The genes targeted by the differentially expressed miRNA were significantly enriched in various pathways, such as the HIF-1 signaling pathway, ErbB signaling pathway, TNF signaling pathway, synaptic vesicle cycle and inositol phosphate metabolism pathway.Real-time quantitative PCR results confirmed that the expression of common differentially expressed miRNA among 3 sample groups (bta-miR-677 and bta-miR-1246) were consistent with RNA-Seq results. 【Conclusion】 Calcium ions indirectly affected the inflammatory response in bovine mammary epithelial cells by regulating the expression of differentially expressed miRNA, such as bta-miR-11980, bta-miR-1246 and bta-miR-677.

Key words: lipopolysaccharide; calcium ion; bovine mammary epithelial cells; miRNA expression profile

中图分类号:

S852.32

许浩天, 于月通, 李静, 马志远, 杨彬, 王泽坤, 陀海欣, 齐萌. 脂多糖与钙离子共处理的牛乳腺上皮细胞miRNA表达谱的比较分析[J]. 中国畜牧兽医, 2024, 51(9): 4066-4079.

XU Haotian, YU Yuetong, LI Jing, MA Zhiyuan, YANG Bin, WANG Zekun, TUO Haixin, QI Meng. Comparative Analysis of miRNA Expression Profiles in Bovine Mammary Epithelial Cells Treated with Lipopolysaccharide and Calcium Ions[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 4066-4079.

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