牙鲆miR-7133和PC-3p-9227调控lamp2在抗海豚链球菌侵染中的作用机理研究

Lysosomes play important roles in the regulation of host immune response, however, the mechanisms need to be further studied. We had performed the miRNA-mRNA co-sequencing in Japanese flounder challenged by Streptococcus iniae and found that the expressions of miR-7133 and PC-3p-9227, were significantly decreased compared to the health group, both of the miRNAs target to the lamp2 (lysosome membrane protein 2) gene in Japanese flounder, and there is a negative regulation relationship between the miRNAs and lamp2. The two miRNAs play a potentially important role in regulating the immune response involved by lysosomes. Based on these preliminary results, we will pursue the function and mechanism of lamp2 regulated by miR-7133 and PC-3p-9227 in Japanese flounder, clarify their temporal and spatial expression patterns, study the effects of miR-7133, PC-3p-9227 and lamp2 on Japanese flounder responding to Streptococcus iniae infection via in vivo and vitro experiments by using methods including overexpression and RNAi, and further explore the mechanism of miRNA regulating the immune response involved by lysosomes. Our results will deepen our understanding of the regulated mechanism of miRNA in fish and provide guidance for the prevention and control of Streptococcus iniae diseases.

溶酶体在抗感染免疫过程中有重要作用，但其调控机理还有待于进一步研究。申请人通过miRNA-mRNA共测序分析发现，牙鲆感染海豚链球菌后，miR-7133和PC-3p-9227显著低表达，且均靶标于溶酶体膜蛋白基因lamp2，它们与lamp2之间是负调控关系，这两个miRNAs在调控溶酶体参与的免疫应答中有潜在的重要作用。基于前期的研究结果，本项目将继续聚焦于miR-7133、PC-3p-9227和lamp2的功能与调控机制，阐明它们的时空表达特征和调控模式；进一步利用RNA干扰、基因过量表达等技术，通过体内、体外实验明确miR-7133和PC-3p-9227及lamp2在牙鲆抗海豚链球菌侵染中的作用，同时进一步解析miRNA调控溶酶体参与的免疫应答。研究结果将加深我们对鱼类miRNA作用机制的理解，同时为海豚链球菌病的防控提供理论参考。

pol-miR-7133和pol-miR-3p-9227均预测共同调控一个溶酶体膜蛋白基因PoLAMP2-3 (an isoform 3 of flounder lysosome-associated membrane protein 2)。研究发现，pol-miR-7133和pol-miR-3p-9227可通过与PoLAMP2-3的3’非翻译区 (3’-UTR)结合来抑制PoLAMP2-3的转录表达。体内体外实验均表明，PoLAMP2-3的表达与pol-miR-7133和pol-miR-3p-9227的表达呈现负调控关系。目前为止，鱼类的LAMP2尚未见报道。我们发现PoLAMP2-3在各组织中均有表达，尤其肝脏中的表达量最高。序列分析发现，PoLAMP2-3是一个酸性的跨膜蛋白，定位于溶酶体、细胞膜和细胞核。体外研究发现，过表达pol-miR-7133和pol-miR-3p-9227或干扰PoLAMP2-3能够促进对牙鲆鳃细胞系（FG cells）感染。与此相一致的是，鱼体内敲降pol-miR-7133和pol-miR-3p-9227或过表达PoLAMP2-3，能够抑制海豚链球菌S. iniae的感染，而敲降PoLAMP2-3则有利于S. iniae的感染。这些结果表明PoLAMP2-3 具有抗S. iniae感染作用。另外，S. iniae感染引起FG cells出现自噬和拉网式病变症状，并且溶酶体的数量显著上升。在过表达pol-miR-7133和pol-miR-3p-9227或干扰PoLAMP2-3的FG cells感染S. iniae后，溶酶体的数量减少，表明受pol-miR-7133和pol-miR-3p-9227调控的PoLAMP2-3可能与溶酶体的生成相关。

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