Research on effect of hypoxia stress on liver tissue of zebrafish (Danio rerio) based on transcriptomics technology
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摘要: 为探究鱼类响应低氧胁迫的调控机制，将1月龄的野生型斑马鱼 (Danio rerio) 在1.5 mg·L−1的低氧浓度下胁迫2个月后，对其肝脏组织进行转录组测序比较分析。对常氧与低氧组的3 270个差异基因进行KEGG分析，主要富集于细胞增殖、脂质代谢、糖类代谢和氨基酸代谢等通路。其中，上调的1 864个基因主要与细胞增殖相关，下调的1 406个基因主要参与脂质代谢。对差异基因进行GO富集分析，发现铁离子束 (Iron ion banding) 功能差异显著。对铁代谢相关基因的表达量进行分析，发现铁离子储存相关基因fthl28和fthl31变化显著，提示在低氧胁迫下斑马鱼肝脏 (Zebrafish liver, ZFL) 组织中铁离子含量发生显著变化。利用ZFL细胞进行体外验证实验，将ZFL细胞进行0.1% (体积分数) O2低氧胁迫，发现随着胁迫时间的延长，ZFL细胞的成活率降低，且细胞中与铁代谢相关基因和铁蛋白 (Ferritin) 的表达均显著降低。综上所述，铁代谢调节是低氧胁迫下的重要响应过程，低氧会导致细胞内铁代谢紊乱，延长低氧时间会形成新的铁稳态。研究结果为探究鱼类的低氧适应机制提供了理论基础和参考。Abstract: To explore the regulatory mechanism of fish response to hypoxia stress, we stressed 1-month-old wild zebrafish (Danio rerio) by hypoxia to 1.5 mg·L−1 for 2 months, and then investigated the liver tissues by transcriptome sequencing and comparative analysis. According to the KEGG analysis performed on 3 270 differential genes in normoxia and hypoxia groups, the genes mainly enriched in pathways such as cell proliferation, lipid metabolism, carbohydrate metabolism and amino acid metabolism. Among them, the up-regulated 1 864 genes were mainly related to cell proliferation, while the down-regulated 1 406 genes were mainly involved in lipid metabolism. We performed a GO enrichment analysis on the differential genes, and found that the function of iron ion banding was significantly different. Based on the analysis of expression of iron metabolism-related genes, the iron ion storage related genes fthl28 and fthl31 changed significantly, which suggests that the iron ion content in zebrafish liver (ZFL) tissue changes significantly under hypoxic stress. Moreover, we conducted the in vitro validation experiments by using ZFL cells which were subjected to 0.1% (Volume fraction) O2 hypoxia stress. The results show that with the prolongation of hypoxia stress time, the survival rate of ZFL cells decreased, and the expression of iron metabolism-related genes and ferritin in the cells decreased significantly. In conclusion, iron metabolism regulation is an important response process under hypoxia stress. Hypoxia may lead to disturbance of intracellular iron metabolism, and prolonging hypoxia time will form a new iron homeostasis. The study provides a theoretical basis and references for exploring the hypoxia adaptation mechanism of fish.
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Key words:
- Danio rerio /
- Transcriptome /
- Hypoxic stress /
- Liver /
- Iron metabolism
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图 1 低氧胁迫下肝脏组织KEGG Pathway富集分布
Figure 1. Enrichment distribution of KEGG Pathway in liver tissue under hypoxia stress
图 2 低氧胁迫肝脏组织差异基因KEGG Pathway富集分布
注：a. 上调基因 KEGG Pathway 富集分布；b. 下调基因 KEGG Pathway 富集分布。
Figure 2. Enrichment and distribution of differential gene KEGG Pathway in liver tissue under hypoxia stress
Note: a. Up-regulated gene KEGG Pathway enrichment distribution; b. Down-regulated gene KEGG Pathway enrichment distribution.
图 3 低氧胁迫后肝脏GO富集分布
Figure 3. Distribution of GO enrichment in liver after hypoxia stress
图 4 低氧胁迫后斑马鱼肝脏组织中与铁代谢相关基因的表达量分析
注：a. 铁代谢相关基因的相对表达量热图；b. 5 个铁代谢相关基因的 RT-qPCR 和 RNA-seq 相对表达量的对比分析。
Figure 4. Analysis of expression levels of genes related to iron metabolism in zebrafish liver tissues after hypoxia stress
Note: a. Heat map of relative expression of iron metabolism relative genes; b. Comparative analysis of relative expression of five iron metabolism related genes of RT-qPCR and RNA-seq.
图 5 低氧胁迫ZFL细胞及细胞成活率、铁代谢相关基因与铁蛋白鉴定
注：*. 与低氧组 (0.1% O2) 相比在 P<0.05 水平存在差异；**. 在 P<0.01 水平差异显著；***. 在P<0.001 水平差异极显著。a. 0.1% O2 胁迫3 d 与常氧条件下 ZFL 细胞存活量对比；b. 0.1% O2 胁迫下 ZFL 细胞活性检测；c. 0.1% O2胁迫 3 d 与常氧条件下 ZFL 细胞中铁代谢相关基因表达量分析；d. 0.1% O2 胁迫后铁蛋白表达量与常氧下对比。
Figure 5. Identification of ZFL cells and cell survival rate, iron metabolism-related genes and Ferritin protein under hypoxia stress
Note: *. Difference (P<0.05) compared with the hypoxia group (0.1% O2); **. Siginificant difference at P<0.01 level; ***. Very significant difference at P<0.001 level. a. Comparison of the viable cell mass of ZFL cells under 0.1% O2 stress for 3 d and normoxia condition; b. Detection of ZFL cell viability under 0.1% O2 stress; c. Analysis of the expression of genes related to iron metabolism in ZFL cells under 0.1% O2 stress for 3 d and normoxia condition; d. Comparison of Ferritin protein expression after 0.1% O2 stress and normoxia condition.
表 1 引物信息
Table 1. Primer information
引物名
Primer name引物序列 (5'—3')
Primer sequences (5'–3')基因ID
Gene IDtfa-F AGCAGCAGACATTGAGTGTC 30255 tfa-R TTTGCTCCATCTACTGTAAC tfr2-F AGCAGTTTACCTCACACTGAC 494476 tfr 2-R AGGAATGTTGTCCGGCTCG fth27-F TGCGAGGCTTTGATCAACAAG 436651 fth27-R TGGCAAATCCAGGAAGAGCC fth28-F AAGATGATCAATCTGGAGC 100006523 fth28-R TTGAAGAACTTGGCAAATCC fth31-F AGGCTGCGATCAACAAGATG 553552 fth31-R AGGAAGAGCCACATCGTC fp n-F ACATGCCCTCTCGACATGG 58153 fpn-R AGGAGTAAACTATTGCCATACAG actin-F TGTCCCTGTATGCCTCTGGT 57934 actin-R AAGTCCAGACGGAGGATG 
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