MicroRNA-515通过靶向整合素β4调控大菱鲆肠道抗菌机制研究

Intestinal mucosa is an important immune barrier in fish, mainly by physiological shedding of intestinal epithelial cells to defend against pathogen invasion. miRNAs play significant roles in intestinal immunity, but their function in fish intestinal mucosal immunity remains largely undetermined. miR-515 is predicted to target integrin beta 4 (itgb4), which is involved in the shedding of intestinal epithelial cells. Moreover, high-throughput sequencing data show that the expression of miR-515 is upregulated in intestinal tissues of turbot resistant to vibrio anguillariae, a kind of pathogenic enterobacteria that severely hazard the aquaculture of turbot. Based on these results, we hypothesize that miR-515 plays a role in the physiological shedding of intestinal epithelial cells after pathogen infection by targeting itgb4. Our previous data confirmed the expression of miR-515 in the intestinal tissues of turbot infected with vibrio anguillaris and identified its target sequence in the 3’UTR of itgb4. Further investigation is required to validate the targeting relation between miR-515 and itgb4, and uncover the role of miR-515 in the shedding of intestinal epithelial cells and related immune pathways. Lessons learned from studying the molecular mechanisms of intestinal immunity in fish will lay important foundations for prevention and control of diseases in turbot.

肠道黏膜是鱼类肠道重要的免疫屏障，肠道上皮细胞生理脱落是肠道黏膜抵御外来病原菌入侵的重要防御机制。miRNA在肠道免疫反应中扮演着重要的角色，但目前缺乏miRNA在鱼类肠道黏膜免疫调控中的相关研究。通过高通量测序发现，在严重危害大菱鲆健康养殖的肠道致病菌—鳗弧菌感染后，预测靶向上皮细胞脱落相关基因itgb4的miR-515，在鳗弧菌耐受的大菱鲆肠道中表达量升高。我们推测，miR-515或许通过靶向itgb4参与病原菌感染后肠道上皮细胞的生理脱落过程。本项目前期验证了miR-515在鳗弧菌感染大菱鲆肠道组织中的表达量，在itgb4 3’UTR中发现了miR-515的靶序列，并且初步证明了miR-515对itgb4的靶向关系。本项目将进一步明确miR-515对itgb4的靶向关系，研究miR-515通过靶向itgb4对肠上皮细胞脱落的影响和参与调控的免疫相关通路，为大菱鲆疾病防控提供理论指导。

肠道上皮细胞生理脱落（主动脱落）是肠道黏膜抵御外来病原菌入侵的重要防御机制。Itgb4在肠道上皮细胞与基底膜牢固粘附中发挥重要作用，miRNA或许通过靶向itgb4来参与病原菌感染后肠道上皮细胞的生理脱落过程。根据项目计划，通过双荧光素酶实验验证了miRNA与itgb4的靶向关系，将miRNA注射到大菱鲆肠道组织中引起itgb4表达的显著下调和肠道上皮细胞的大量脱落。但是对于后续miRNA通过靶向itgb4如何参与病原菌感染后的鱼类肠道上皮细胞生理脱落，仍需确定病原菌通过itgb4介导肠道上皮细胞脱落的机制。因此，将我们研究重点调整为itgb4在肠道上皮细胞脱落中的作用。大菱鲆itgb4高表达于黏膜组织皮肤、鳃和肠道中，在鳗弧菌感染早期，大菱鲆肠道组织中itgb4表达显著下调，并引发了肠道上皮细胞大量的生理脱落。分离的肠道上皮细胞相对于其它肠道细胞高表达itgb4基因，通过亚细胞定位结果验证了大菱鲆itgb4表达于细胞膜上。将itgb4的siRNA注射到大菱鲆肠道组织中引发了上皮细胞的大量脱落并导致了免疫相关基因的表达上调和其他细胞粘附相关基因的表达下调。构建了大菱鲆肠道细胞系（SMI），并通过其构建了itgb4稳定干扰的细胞系，在受到poly(I:C)刺激后，itgb4稳定干扰后的SMI相比正常的SMI凋亡增加；构建了itgb4的斑马鱼敲除模型，F2代itgb4敲除的纯合品系（itgb4-/-）至多存活12天，通过HE染色结果发现itgb4-/-斑马鱼显示出肠道上皮细胞的不稳定性。以上结果均证实了itgb4在肠道上皮层细胞稳定粘附中的作用。本项目开展过程中构建的大菱鲆肠道细胞系和itgb4纯合突变的斑马鱼品系为深入研究鱼类肠道上皮细胞的生理脱落机制打下基础。