MicroRNA在镉致鲤鱼免疫反应中的作用机制

Cadmium is a heavy metal that commonly found in the water throughout the world. Cadmium has a long biological half-life, which can accumulate to significant levels in the body, and lead to detrimental effects in animals and human eventually. Excess intake of cadmium will cause the immune response; induce innate immunity and adaptive immunity of fish. However, there is no clearly about the pathway and mechanism of immune response in fish induced by cadmium. This project will screen the target organ of cadmium by detecting different levels of cadmium and effects on nonspecific immune in the tissues in fish exposed to cadmium; then, using high-throughput sequencing found different microRNAs (miRNAs) and their target genes within target organ between control and test groups; furthermore, verify the gene expression correspondly by qRT-PCR. Moreover, we will analysis the the correlations between miRNAs and their target genes. Lastly, using miRNA agomir and miRNA antagomir, injection of fish to detect miRNAs and their target genes expression, validate correlations between miRNAs and their target genes again. Meanwhile, obtain primary cells for culture to look for binding sites of miRNAs and their target genes through dual-luciferase reporter gene assay.

镉是水体中重金属污染最常见的一种，其极易通过食物链在机体内富集，且半衰期很长，会对机体产生严重危害。水体中镉含量超标引起鱼类的免疫响应，影响鱼的先天性免疫和获得性免疫。对于镉导致鱼类免疫反应的途径及其作用机理，目前还没有阐述清楚。本项目通过对鲤鱼进行镉暴露，检测镉在鲤鱼体内的蓄积及对内脏非特异性免疫指标的影响，确定镉作用的靶器官。检测暴露后靶器官miRNA的表达差异，并通过qRT-PCR验证差异显著的miRNA，对和测序结果一致的miRNA进行靶基因预测。提取靶器官的mRNA，检测靶基因mRNA的表达，确定miRNA和其靶基因的对应关系，合成miRNA的激动剂miRNA agomir和抑制剂miRNA antagomir，对鱼进行注射，检测miRNA和其靶基因的表达，再次验证miRNA和靶基因的靶向关系。培养鲤鱼的靶器官原代细胞，利用双荧光素酶报告基因系统确定miRNA与靶基因的结合位点。

本项目对鲤进行不同浓度镉暴露，检测了镉在鲤不同组织蓄积量，对组织的非特异性免疫酶活性进行测定，制作组织切片，使用光学显微镜进行观察，最终发现肾脏是镉致鲤免疫作用的靶器官。通过高通量测序发现 0.5 mg/L Cd2+ 导致9个miRNAs的表达发生显著变化，包括miR-217、miR-205和7个novel-miRNAs，利用RT-PCR对9个表达差异显著的miRNAs进行验证，发现除了miR-205之外，其余8个miRNAs的表达趋势与高通量测序结果一致。合成miR-217的激动剂miR-217 agomir，给鲤腹腔注射后，发现肾脏内miR-217含量是对照组的54倍，同时检测肾脏中AKP和ACP活性，发现miR-217 agomir显著改变了肾脏中AKP和ACP的活性。通过预测miRNA的靶基因，并检测注射miR-217 agomir后靶基因的表达，与镉暴露组靶基因的表达量对比，发现miR-217是通过抑制靶基因SIRT1的表达参与镉致鲤的免疫反应。同时检测镉暴露组免疫相关miRNAs含量，发现miR-155和miR-181a参与镉致鲤的免疫反应，给鲤腹腔注射miR-155 agomir、miR-181a antagomir后，检测其靶基因和免疫相关基因表达，与镉暴露组的表达量对比，发现miR-155和miR-181a通过调节HO-1的表达在镉致鲤的免疫响应中发挥作用。本项目明确了miR-217、miR-155和miR-181a参与镉致鲤免疫响应的作用通路，研究结果对于阐述miRNAs如何参与镉致鲤的免疫应答具有重要意义。