Table_4_Sea Urchins in Acute High Temperature and Low Oxygen Environments: The Regulatory Role of microRNAs in Response to Environmental Stress.xlsx

Strongylocentrotus intermedius is an economically valuable sea urchin species in China. However, its growth and survival are severely constrained by ocean warming and the hypoxia that often accompanies high water temperatures. MicroRNAs (miRNAs) are important regulators of gene expression in response to environmental change. In this study, high-throughput RNA sequencing was used to investigate changes in miRNA expression in S. intermedius under heat (25°C), hypoxia (2 mg/L O2), and combined heat and hypoxia stresses. Twelve small RNAs libraries were constructed and 17, 14, and 23 differentially expressed miRNAs (DEMs) were identified in the heat, hypoxia, and combined stress groups (P<0.05), respectively. Gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway functional analyses of putative target genes of the DEMs suggested that these miRNAs were important in basal metabolism, apoptosis, oxidative stress, and immune-related pathways. By co-analysis with published transcriptome data, key DEMs (miR-193, miR-184, miR-133, miR-125, miR-2008) and their key target genes (EGF3, ABCB4, CYCL, PAN2, CALN) were identified. Quantitative real-time PCR analysis of the expression of 10 DEMs and their key target genes confirmed the RNA sequencing results. These results provide information on gene expression regulation of the molecular mechanisms underlying the response of S. intermedius to multi-cause environmental stresses.

中间球海胆（Strongylocentrotus intermedius）是我国一种具有重要经济价值的海胆物种。然而，海洋变暖以及伴随高温出现的低氧环境严重制约了其生长与存活。微小RNA（microRNAs，miRNAs）是响应环境变化的关键基因表达调控因子。本研究采用高通量RNA测序技术，探究了中间球海胆在高温（25℃）、低氧（2 mg/L O₂）以及高温与低氧联合胁迫下的miRNA表达变化。共构建12个小RNA文库，在高温、低氧及联合胁迫组中分别鉴定出17、14和23个差异表达微小RNA（differentially expressed miRNAs，DEMs），且所有结果均满足P<0.05的显著性阈值。对DEMs的潜在靶基因开展基因本体（Gene Ontology，GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路功能富集分析，结果表明这些miRNAs在基础代谢、细胞凋亡、氧化应激及免疫相关通路中发挥重要调控功能。结合已发表的转录组数据进行联合分析，本研究鉴定出关键DEMs：miR-193、miR-184、miR-133、miR-125、miR-2008，及其对应的关键靶基因：EGF3、ABCB4、CYCL、PAN2、CALN。通过实时荧光定量PCR（quantitative real-time PCR）对10个DEMs及其关键靶基因的表达水平进行验证，结果证实了RNA测序的分析结论。本研究结果为解析中间球海胆响应多因素环境胁迫的分子机制提供了基因表达调控层面的科学依据。