MiR-155靶向踝蛋白抑制柱状黄杆菌诱导EPC细胞凋亡的机制研究

The infection of Flavobacterium columnare (FC) has caused great economic loss in fish culture worldwide. However, up to date, there is not effective prevention strategies against FC infection available. MicroRNA plays a key role in bacterial infections. In the previous study, we observed that both FC infection and incubation of extracellular polysaccharide (EPS ) derived from FC was able to significantly up-regulate the expression of miR-155 in Epithelioma Papulosum Cyprini cells (EPC), resulting in the cellular apoptosis. In addition, the target gene prediction results show that talin-1 is the target gene of miR-155. Transfection of miR-155 mimic was able to down-regulate the expression of talin-1 and inhibited the cellular apoptosis induced by the EPS. However, the mechanism underlying the inhibition remains enigmatic. Based on the above results, we will perform the following experiments in the current proposal: (1). Characterize the three classic pathways of apoptosis induced by the EPS.(2). The effects of knock down or over-expression of the talin-1 protein on the three classic pathways of apoptosis induced by the EPS. The results will decipher the mechanism of the inhibition of EPS–induced apoptosis by miR-155 via targeting the talin-1 gene in EPC cells, and will pave a new way for the development of prevention strategies against the infection of FC.

柱状黄杆菌感染给鱼类养殖造成重大经济损失，目前尚无高效防控措施。MicroRNA在细菌的感染中发挥重要作用。我们前期的研究发现柱状黄杆菌感染或者其胞外多糖孵育都能显著提高鲤上皮瘤细胞（EPC）miR-155的表达，并导致EPC细胞的凋亡。通过靶基因分析，miR-155靶向鲤踝蛋白基因(talin-1)。转染miR-155类似物能降低踝蛋白的表达，并可抑制胞外多糖诱导的EPC细胞凋亡,但其机制尚不清楚。据此，本项目拟研究：（1）柱状黄杆菌胞外多糖诱导三个经典细胞凋亡途径及其相关效应蛋白的变化；（2）敲降或过度表达鲤踝蛋白对胞外多糖诱导的三个经典细胞凋亡途径及其相关效应蛋白的变化。研究结果将阐明miR-155通过靶向鲤踝蛋白来调控柱状黄杆菌胞外多糖导致细胞凋亡的机制，为防控该菌的感染提供新思路。

柱状黄杆菌感染给水产养殖造成重大经济损失，目的尚无高效的防治措施。柱状黄杆菌胞外多糖孵育能够诱导细胞发生凋亡。体内和体外实验表明柱状黄杆菌胞外多糖诱导的凋亡是通过caspase-3依赖的方式,经由死亡受体途径诱导细胞凋亡。.柱状黄杆菌胞外多糖能提高鲤上皮瘤细胞（EPC）miR-155的表达，转染miR-155类似物能降低踝蛋白表达，并可抑制胞外多糖诱导的细胞凋亡。采用EMSA和Western-blot两种方法确定踝蛋白talin-1是miR-155的靶蛋白，高表达的miR-155（转染24h可达600倍）通过抑制踝蛋白表达来调控细胞凋亡。.柱状黄杆菌胞外多糖孵育EPC细胞发现踝蛋白表达量先升高再下降，然后慢慢恢复到正常水平。在凋亡发生的早期阶段同时检测到踝蛋白的片段化（在踝蛋白的C端两条大小在150-250kd的条带）。构建踝蛋白C端真核表达质粒（talin-p120）转染到EPC细胞，过表达的踝蛋白延迟了细胞凋亡的发生（由8-24h延迟到48-96h发生凋亡），证明踝蛋白通过调节表达量和片段化直接参与胞外多糖诱导的凋亡调控；本研究还发现胞外多糖感染导致细胞肌动蛋白表达量的下调，诱导细胞骨架发生改变和移动。踝蛋白与肌动蛋白共同参与调节柱状黄杆菌胞外多糖致病机制。