Abstract：
MicroRNAs play crucial roles in various biological processes, including but not limited to differentiation, development, disease, and immunity. However, their immunoregulatory roles in half-smooth tongue sole are lacking. Our previous studies indicated that miR-722 could target C5aR1 to modulate the complement pathway to alleviate inflammatory response and even affect the mortality after the bacterial infection with Vibrio anguillarum. Driven by the purpose of revealing the underlying mechanisms, in this study, we investigated the effects of miR-722 on the gene expression and alternative splicing (AS) in the liver of half-smooth tongue sole after Vibrio anguillarum infection, with the approach of miR-722 overexpression/silencing and subsequent RNA-seq. Among the different comparisons, the I group (miR-722 inhibitor and V. anguillarum) versus blank control (PBS) exhibited the highest number of differentially expressed genes (DEGs), suggesting that the immune response was overactivated after inhibiting the miR-722. In addition, enrichment analyses were performed to reveal the functions of DEGs and differential AS (DAS) genes, reflecting the enrichment of RNA splicing and immune-related pathways including NF-κB and T cell receptor signaling pathway. Comparing the M group (miR-722 mimic and V. anguillarum) with the negative control (random sequence and V. anguillarum), two immune-related genes, cd48 and mapk8, were differentially expressed, of which mapk8 was also differentially spliced, indicating their importance in the immune response. Furthermore, representative gene analysis was performed, suggesting their corresponding functional changes due to AS. To verify the RNA-seq data, quantitative real-time PCR was employed with twenty pairs of primers for DEGs and DAS events. Overall, our results demonstrated that miR-722 could mediate the transcriptome-wide changes of gene expression and AS in half-smooth tongue sole, and provided insights into the regulatory role of miR-722 in immune responses, laying the foundation for further functional analyses and practical applications in aquaculture.
摘要：
MicroRNAs在各种生物过程中发挥关键作用，包括但不限于差异化，发展,疾病,和豁免权。然而,它们在半光滑舌底中缺乏免疫调节作用。我们之前的研究表明，miR-722可以靶向C5aR1调节补体途径，减轻炎症反应，甚至影响弧菌感染后的死亡率。在揭示潜在机制的目的的驱动下，在这项研究中，我们研究了miR-722对半光滑舌根感染后肝脏中基因表达和选择性剪接（AS）的影响，与miR-722过表达/沉默和随后的RNA-seq的方法。在不同的比较中，I组（miR-722抑制剂和V.anguillarum）相对于空白对照（PBS）表现出最高数量的差异表达基因（DEGs），提示抑制miR-722后免疫反应过度激活。此外,进行了富集分析，以揭示DEGs和差异AS（DAS）基因的功能，反映了RNA剪接和免疫相关通路的富集，包括NF-κB和T细胞受体信号通路。比较M组(miR-722模拟物和anguillarum弧菌)与阴性对照(随机序列和anguillarum弧菌),两个免疫相关基因，cd48和mapk8差异表达，其中mapk8也被差分拼接，表明它们在免疫反应中的重要性。此外，进行了代表性基因分析，提示它们由于AS而相应的功能变化。为了验证RNA-seq数据，使用二十对引物对DEG和DAS事件进行定量实时PCR。总的来说,我们的结果表明，miR-722可以介导全转录组的基因表达和AS在半滑舌底的变化，并提供了对miR-722在免疫反应中的调节作用的见解，为进一步的功能分析和水产养殖的实际应用奠定基础。
