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岳小真, 常娇娇, 常新月, 李槿年. 抗菌肽Hepcidin诱导草鱼肾细胞miRNA表达分析[J]. 水生生物学报, 2023, 47(11): 1827-1837. DOI: 10.7541/2023.2023.0038
引用本文: 岳小真, 常娇娇, 常新月, 李槿年. 抗菌肽Hepcidin诱导草鱼肾细胞miRNA表达分析[J]. 水生生物学报, 2023, 47(11): 1827-1837. DOI: 10.7541/2023.2023.0038
YUE Xiao-Zhen, CHANG Jiao-Jiao, CHANG Xin-Yue, LI Jin-Nian. ANALYSIS OF miRNA EXPRESSION PROFILES IN CTENOPHARYNGODON IDELLA KIDNEY CELLS INDUCED BY THE ANTIBACTERIAL PEPTIDE HEPCIDIN[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(11): 1827-1837. DOI: 10.7541/2023.2023.0038
Citation: YUE Xiao-Zhen, CHANG Jiao-Jiao, CHANG Xin-Yue, LI Jin-Nian. ANALYSIS OF miRNA EXPRESSION PROFILES IN CTENOPHARYNGODON IDELLA KIDNEY CELLS INDUCED BY THE ANTIBACTERIAL PEPTIDE HEPCIDIN[J]. ACTA HYDROBIOLOGICA SINICA, 2023, 47(11): 1827-1837. DOI: 10.7541/2023.2023.0038

抗菌肽Hepcidin诱导草鱼肾细胞miRNA表达分析

ANALYSIS OF miRNA EXPRESSION PROFILES IN CTENOPHARYNGODON IDELLA KIDNEY CELLS INDUCED BY THE ANTIBACTERIAL PEPTIDE HEPCIDIN

  • 摘要: 为探讨草鱼抗菌肽Hepcidin(CiHep)过表达前后草鱼肾细胞系(Ctenopharyngodon idella kidney, CIK)miRNA表达谱变化及其分子机制, 将构建的重组表达质粒pmCherry-N1-Cihep转染CIK细胞, 通过检测CiHep基因转录水平及融合蛋白mCherry-CiHep荧光强度, 筛选其最佳过表达时间。在最佳过表达时间点收集CIK细胞样本, 进一步利用高通量测序和qPCR技术对差异表达miRNA表达谱进行分析。结果显示, CiHep基因在CIK细胞中最佳过表达时间为转染后72h。从构建的2个sRNA文库中分别鉴定到1850与2013种已知miRNAs, 并发现1266与1347种新miRNAs。与对照组相比, 过表达组共筛选到460个DEmiRNAs, 其中392个显著上调, 68个显著下调。对5150个DEmiRNA靶基因进行GO注释和KEGG通路分析, 发现436个靶基因得到GO注释, 主要参与细胞进程、生物学调节和单细胞生物进程等生物学过程; 157个靶基因富集于54条KEGG通路。miRNA-mRNA-免疫互作网络分析显示, 29条DEmiRNA和23个靶基因潜在介导CiHep参与C型凝集素受体、PI3K-Akt、NOD样受体等13个免疫相关信号通路。12个DEmiRNAs的qPCR验证结果与高通量测序结果一致。研究解析了CiHep过表达前后CIK细胞miRNA差异表达谱特征, 明晰pma-miR-199b-5p、dre-miR-15a-5p、novel_miR_317和novel_miR_536在免疫调控网络中发挥重要作用, 为深入探究鱼类抗菌肽诱导miRNA免疫调节的分子机制奠定了基础。

     

    Abstract: To investigate the changes of miRNA expression profile of Ctenopharyngodon idella kidney cell line (CIK) before and after overexpression of the Ctenopharyngodon idella antimicrobial peptide Hepcidin (CiHep), and its molecular mechanism, the constructed recombinant expression plasmid pmCherry-N1-Cihep was transfected into CIK cells, and its optimal overexpression time was screened by detecting the transcription level of the gene and observing the fluorescence intensity of fusion proteinm Cherry-CiHep. CIK cell samples were collected at the optimal overexpression time point, and further analyzed for differentially miRNA expression profiles using high-throughput sequencing and qPCR techniques. The results showed that the optimal overexpression time of CiHep gene in CIK cells was 72h after transfection. 1850 and 2013 known miRNAs were identified as well as 1266 and 1347 new miRNAs were found from the two constructed sRNA libraries, respectively. Compared with the control group, 460 DEmiRNAs were screened in the overexpression group, among which 392 were significantly up-regulated and 68 were significantly down-regulated. GO annotation and KEGG pathway analysis of 5150 DEmiRNA target genes revealed that 436 target genes were GO annotated and mainly involved in biological processes such as cellular processes, biological regulation and single cell biological processes. 157 target genes were enriched in 54 KEGG pathways. miRNA-mRNA-immune interaction network analysis revealed that 29 DEmiRNAs and 23 target genes potentially mediated CiHep involvement in 13 immune-related signaling pathways including C-type lectin receptor, PI3K-Akt, and NOD-like receptor. The qPCR validation results of 12 DEmiRNAs were consistent with high-throughput sequencing results. The characteristics of miRNA expression profiles of CIK cells before and after CiHep overexpression were resolved in the study, and clarified pma-miR-199b-5p, dre-miR-15a-5p, novel_miR_317 and novel_miR_536 played a potentially important role in the immune regulatory network, which laid the foundation for an in-depth investigation of the molecular mechanisms of miRNA immune regulation induced by antimicrobial peptides in fish.

     

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