猪miR-192对DLG5和ALCAM基因的靶向作用关系验证  

Validation of the Targeting Effects of Swine(Sus scrofa) miR-192 on the DLG5 and ALCAM Genes

作　　者：孙丽[1] 吴森[1] 吴嘉韵 赵呈祥[1] 吴圣龙[1,2] 包文斌[1,2] 

机构地区：[1]扬州大学动物科学与技术学院/江苏省动物遗传繁育与分子设计重点实验室,扬州225009 [2]扬州大学教育部农业与农产品安全国际合作联合实验室,扬州225009

出　　处：《农业生物技术学报》2017年第9期1451-1459,共9页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31372285和No.31572360);江苏省科技支撑计划(No.BE2014357和No.BE2015329)

摘　　要：在生理病理过程中,micro RNAs(miRNAs)通过作用于相应靶基因发挥着重要的作用。通过高通量测序结合荧光定量实验验证,本课题组前期初步确定miR-192靶向调控Discs大同源物(discs large homolog 5,DLG5)和活化白细胞黏附分子(activated leukocyte cell adhesion molecule,ALCAM)基因在断奶仔猪抗大肠杆菌(Escherichia coli)感染过程中发挥了重要的作用。本研究采用双荧光素酶报告系统和Western blot方法,验证DLG5和ALCAM基因是否受到miR-192的特异性调控。构建含有靶基因位点的荧光素酶报告基因重组载体,与PRL-TK和miRNA-192模拟物mimics、inhibitor或阴性对照共转染293细胞,24 h后收集细胞检测荧光素酶活性和靶基因蛋白表达变化。同时检测3种大肠杆菌感染肠上皮细胞(intestinal epithelial cells,IPEC-J2)后靶基因的表达变化。本研究成功获得荧光素酶报告基因重组载体,荧光结果显示,miRNA-192 mimics显著抑制2个报告重组载体的荧光素酶活性(P<0.05),而miRNA-192inhibitor则显著促进2个报告重组载体的荧光素酶活性(P<0.05)。同时,miRNA-192模拟物处理组靶基因DLG5和ALCAM蛋白水平极显著低于阴性对照组,再次验证了上述结果。3种大肠杆菌感染后,2个靶基因的表达均显著或极显著上升。由结果可知,猪miR-192对DLG5和ALCAM基因具有靶向抑制作用,且DLG5和ALCAM基因的表达确实与大肠杆菌感染有关。本研究结果为miR-192及其靶基因在断奶仔猪抵抗F18大肠杆菌感染过程中的功能和调控机制相关研究提供了一定的实验基础和理论依据,进一步为猪抗大肠杆菌病有效遗传标记的筛选提供了科学依据。Post-weaning diarrhea （PWD） is a very common infectious disease in pig （Sus scrofa） production, which has caused huge losses to the pig industry. MicroRNAs（miRNAs） play an important role in physiological and pathological processes by acting on the corresponding target genes. MiRNAs mainly perform complete or incomplete pairing with 3＇ UTR of target mRNA to degrade target mRNA or inhibit mRNA translation. Afterwards, the transcriptional expression of the target gene is regulated to affect the biological activity of the individual. Our previous studies have screened out miR- 192 which may be important miRNAs for regulating Escherichia coli F 18 infection in weaned piglets by high-throughput sequencing and verification test, and these studies have preliminarily screened out two key target genes discs large homolog 5 （DLG5） and activated leukocyte cell adhesion molecule （ALCAM） which regulate E. coli F 18 infection. In this study, the dual-luciferase reporter system and Western blotting analysis were performed to explore whether DLG5 and ALCAM genes were under the specific regulation of miR- 192. Recombinant plasmids which included miRNA target sites were constructed and were co-transfected with PRL-TK and miRNA-192 mimics, inhibitor or NC into 293 cells （miRNA- 192 mimics and negative control）. Cells were collected for detection of luciferase activity and protein levels after 24 h transfection. Then mRNA expression of two target genes in IPEC-J2 cells infected by E. coli was detected. The 3＇UTR regions of 2 target genes were constructed into the dual-luciferase reporter vector. Then, this study successfully obtained luciferase reporter gene recombinant plasmids. The results of luciferase activity showed that miR-192 mimics could bind to binding sites of target genes and significantly inhibit luciferase activity of ALCAM and DLG5 3＇UTR dual-luciferase reporter recombinant vector （P〈0.05）, and miR-192 inhibitor could significantly enhance luciferase activity of ALCAM and DLG5 3＇UT

关 键 词：猪 miRNA-192 活化白细胞黏附分子基因(ALCAM) Discs大同源物基因(DLG5) 腹泻 

分 类 号：S857.3[农业科学—临床兽医学]