MiR-155靶向talin-1基因调控柱状黄杆菌胞外多糖诱导的EPC细胞凋亡  

MiR-155 regulates apoptosis induced by FC-EPS incubation via targeting the talin-1 gene in EPC

作　　者：王骞 王承德 罗敏意 黎果 车行 林蠡 赵丽娟 WANG Qian;WANG Chengde;LUO Minyi;LI Guo;CHE Hang;LIN Li;ZHAO Lijuan(College of Animal Science and Technology,Zhongkai University of Agriculture and Engineering,Guangzhou 510225,China;Agricultural Service Center of Xiaolan Town,Zhongshan,510230,China;Bailian Aquatic Seedling Company Ltd.of Gaozhou City,Maoming 525200,China)

机构地区：[1]仲恺农业工程学院动物科技学院,广东广州510225 [2]中山市小榄镇农业服务中心,广东中山510230 [3]高州市百联水产种苗有限公司,广东茂名525200

出　　处：《水产学报》2024年第5期24-31,共8页Journal of Fisheries of China

基　　金：国家自然科学基金(31602190);中山市2022年第二批社会公益与基础研究项目(2022B2003);2021年广东省科技创新和乡村振兴战略专项(2021S0082);广东省现代农业产业技术体系创新团队-水产疫病监测与综合防控共性关键技术研发创新团队(2019KJ141)。

摘　　要：为研究miR-155在柱状黄杆菌胞外多糖(FC-EPS)诱导的细胞凋亡中的作用机制,本实验采用RNA干扰(RNAi)技术,通过过表达miR-155和敲降踝蛋白基因(talin-1),皆能抑制FC-EPS诱导的细胞凋亡,并鉴定出talin-1为miR-155的靶蛋白。在FC-EPS诱导细胞凋亡的过程中,踝蛋白水平显著升高,在凋亡发生阶段检测到凋亡执行蛋白Caspase-3的活化,同时检测到2条踝蛋白剪切异构体,大小分别约为200 ku和250 ku;将构建的鲤Talin-1真核表达质粒转染鲤上皮瘤细胞(EPC),过表达的Talin-1延迟且延长了细胞凋亡的进程,并检测到上述2条踝蛋白异构体。然而,当以FC-EPS转染EPC细胞时,Talin-1先下降再恢复到正常水平,细胞不发生凋亡。因此,在FC-EPS诱导细胞凋亡的过程中,细胞通过上调miR-155来调控靶基因talin-1 mRNA和蛋白转录的变化,以及产生Talin-1蛋白异构体来抑制细胞发生凋亡。本研究为有效防治柱状黄杆菌疾病提供新思路。Incubation of Flavobacterium columnare exopolysaccharide(FC-EPS)significantly increases the expression of miR-155 in epithelioma papulosum cyprinid(EPC)cell line,leading to cell apoptosis.In order to study the mechanism of miR-155 in FC-EPS induced apoptosis,RNAi technology was used in this research.It was found that both overexpression of miR-155 and talin-1 knockout could inhibit apoptosis induced by FC-EPS incubation.Then,talin-1 was identified as the target protein of miR-155 by EMSA assay.During the process of apoptosis induced by FC-EPS incubation,the Talin-1 protein was significantly increased,and the activation of apoptosis executioner caspase-3 protein was detected.At the same time,two talin-1 isomers(about 200 ku and 250 ku,respectively)were obtained.Subsequently,the talin-1 gene inserted in plasmid pcDNA3.1 was constructed and transfected into EPC cells,the overexpressed Talin-1 protein delayed and prolonged the process of apoptosis,and two Talin-1 isomers mentioned above were detected.However,apoptosis did not occur when EPC cells were transfected with FC-EPS;on this occasion,the Talin-1 protein in EPC went down at first,then returned to normal level.Hence,in the context of apoptosis induced by FC-EPS incubation,EPC cells reduces apoptosis by regulating the level of mRNA and protein transcription of talin-1 through up-regulation of miR-155 and producing Talin-1 isomers.This paper provides a new idea for effective prevention and treatment of F.columnare diseases.

关 键 词：柱状黄杆菌 MIR-155 talin-1 细胞凋亡 RNA干扰 

分 类 号：Q933[生物学—微生物学] S942[农业科学—水产养殖]