ssc-miR-204靶向调控猪小肠上皮细胞中DLG5基因的表达  

ssc-miR-204 Targeted Regulation the Expression of DLG5 Gene in Intestinal Porcine(Sus scrofa)Epithelial Cells

作　　者：王伟 谢开会 雒瑞瑞 高小莉[1] 王鹏飞[1] 张娟丽 杨姣姣[1] 张博 马艳萍[3] 滚双宝[1,2] WANG Wei;XIE Kai-Hui;LUO Rui-Rui;GAO Xiao-Li;WANG Peng-Fei;ZHANG Juan-Li;YANG Jiao-Jiao;ZHANG Bo;MAYan-Ping;GUN Shuang-Bao(College of Animal Science and Technology,Gansu Agricultural University,Lanzhou 730070,China;Gansu Research Center for Swine Production Engineering and Technology,Lanzhou 730070,China;Gansu Agricultural University Library,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物科学技术学院,兰州730070 [2]甘肃省现代养猪技术工程研究中心,兰州730070 [3]甘肃农业大学图书馆,兰州730070

出　　处：《农业生物技术学报》2021年第6期1083-1093,共11页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(31960646);甘肃省现代农业产业技术体系猪鸡产业(GARS-ZJ-1)。

摘　　要：ssc-miR-204在仔猪(Sus scrofa)感染C型产气荚膜梭菌耐受和易感组中显著差异表达,本研究旨在验证miR-204和Discs大同源物5(discs large homolog 5,DLG5)基因之间的靶向调控关系。利用TargetScan软件预测miR-204与DLG5基因之间的靶向结合位点及保守性;构建DLG5基因3’UTR区野生型及突变型pmirGLO双荧光素酶报告载体,通过荧光素酶活性检测miR-204与DLG5基因之间的靶向关系;进一步在猪小肠上皮细胞(intestine porcine epithelial cells,IPEC-J2)中转染miR-204 mimic和inhibitor,通过qRT-PCR、Western blot和细胞免疫荧光实验,检测miR-204对DLG5表达的调控。结果表明,miR-204成熟体种子区序列与DLG5基因3’UTR第1333~1339位核苷酸序列互补配对,并且在多个物种之间高度保守。通过双酶切和测序鉴定,本研究成功构建pmirGLO-DLG5-3’UTR双荧光素酶报告基因重组载体;荧光素酶活性检测结果显示,miR-204 mimic可以极显著降低DLG5基因的荧光素酶活性(P<0.01),二者具有靶标关系。qRT-PCR、Western blot和细胞免疫荧光结果显示,转染miR-204 mimic后,可以极显著抑制DLG5的表达水平(P<0.01);相反,转染miR-204 inhibitor后,DLG5被显著或极显著上调表达(P<0.05或P<0.01)。miR-204可以靶向结合DLG5基因,miR-204对DLG5的表达具有负调控作用,本研究结果可为进一步在细胞水平验证miR-204和DLG5基因功能提供基础,为miRNAs参与调控C型产气荚膜梭菌性仔猪腹泻提供依据。Based on early study,the high-throughput sequencing found that ssc-miR-204 was significantly differentially expressed in piglets infected with Clostridium perfringens type C in the resistance and susceptibility group.This study aims to verify the targeted regulatory relationship between miR-204 and discs large homolog 5 gene(DLG5).In the present study,the TargetScan software was used to predict the targeted binding sites and conservation between miR-204 and DLG5 gene.The wild-type and mutant-type pmirGLO dual-luciferase reporter vector in the 3’UTR region of DLG5 gene was constructed and the targeting relationship between miR-204 and DLG5 confirmed by dual luciferase activity experiment.Further,the regulation of miR-204 on DLG5 expression was detected by qRT-PCR,Western blot and cell immunofluorescence experiments after transfected with miR-204 mimic and inhibitor in intestine porcine epithelial cells,respectively.The results showed that the miR-204 mature seed region sequence was complementary to the nucleotide sequence of DLG5 at 3’UTR 1333~1339,and was highly conserved among multiple species(such as human(Homo sapiens),pig,chimpanzee(Pan troglodytes),rat(Rattus norvegicus),rabbit(Oryctolagus cuniculus),cattle(Bos taurus),etc).Through double enzyme digestion and sequencing identification,this experiment successfully constructed pmirGLO-DLG5-3’UTR dual luciferase vector;the results of dual luciferase reporter gene experiment showed that miR-204 mimic could significantly reduce the luciferase activity of DLG5 gene(P<0.01),the two had a target relationship.Then,the results of q RT-PCR and Western blot indicated that after transfected with miR-204 mimic,the expression level of DLG5 was significantly inhibited(P<0.01).On the contrary,after transfected with miR-204 inhibitor,the expression level of DLG5 was significantly upregulated(P<0.01).The immunofluorescence results showed that compared with the mimic NC group(89.90±3.818),DLG5 fluorescence intensity was significantly reduced(62.65±3.790,P<0.01)after tra

关 键 词：ssc-miR-204 Discs大同源物5基因(DLG5) 双荧光素酶活性实验 猪小肠上皮细胞(IPEC-J2) 仔猪腹泻 

分 类 号：S828.89[农业科学—畜牧学]