Zhao Xiaomin, Song Xiangjun, Bai Xiaoyuan, Fei Naijiao, Huang Yong, Zhao Zhimin, Du Qian, Zhang Hongling, Zhang Liang, Tong Dewen
College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China.
PeerJ. 2016 Feb 4;4:e1635. doi: 10.7717/peerj.1635. eCollection 2016.
Transmissible gastroenteritis virus (TGEV), belonging to the coronaviridae family, is the key cause of the fatal diarrhea of piglets and results in many pathological processes. microRNAs (miRNAs) play a key role in the regulation of virus-induced apoptosis. During the process of apoptosis induced by TGEV infection in PK-15 cells, the miR-27b is notably down-regulated. Thus, we speculate that miR-27b is involved in regulating the process of apoptosis in PK-15 cells. In this study we demonstrated that the over-expression of miR-27b led to the inhibition of TGEV-induced apoptosis, reduction of Bax protein level, and decrease of caspase-3 and -9 activities. Conversely, silencing of miR-27b by miR-27b inhibitors enhanced apoptosis via up-regulating Bax expression and promoting the activities of caspase-3 and -9 in TGEV-infected cells. Subsequently, the runt-related transcription factor 1 (RUNX1) is a candidate target of miR-27b predicted by bioinformatics search. We further identified that the miR-27b directly bound to the 3' UTR of RUNX1 mRNA and suppressed RUNX1 expression, which indicates RUNX1 is the direct target gene of miR-27b. The over-expression of RUNX1 increased apoptosis and knockdown RUNX1blocked apoptosis of viral-infected cells via regulating Bax expression and the activities of caspase-3 and -9. Our data reveal that miR-27b may repress the mitochondrial pathway of apoptosis by targeting RUNX1, indicating that TGEV may induce apoptosis via down-regulating miR-27b and that miR-27b may act as a target for therapeutic intervention.
传染性胃肠炎病毒(TGEV)属于冠状病毒科,是仔猪致命性腹泻的主要病因,并引发多种病理过程。微小RNA(miRNA)在病毒诱导的细胞凋亡调控中起关键作用。在TGEV感染PK - 15细胞诱导凋亡的过程中,miR - 27b显著下调。因此,我们推测miR - 27b参与调控PK - 15细胞的凋亡过程。在本研究中,我们证明miR - 27b的过表达导致TGEV诱导的凋亡受到抑制,Bax蛋白水平降低,以及caspase - 3和 - 9活性下降。相反,用miR - 27b抑制剂沉默miR - 27b可通过上调Bax表达并促进TGEV感染细胞中caspase - 3和 - 9的活性来增强细胞凋亡。随后,通过生物信息学搜索预测,与 runt 相关的转录因子1(RUNX1)是miR - 27b的候选靶标。我们进一步鉴定出miR - 27b直接与RUNX1 mRNA的3'UTR结合并抑制RUNX1表达,这表明RUNX1是miR - 27b的直接靶基因。RUNX1的过表达增加细胞凋亡,而敲低RUNX1可通过调节Bax表达以及caspase - 3和 - 9的活性来阻止病毒感染细胞的凋亡。我们的数据表明,miR - 27b可能通过靶向RUNX1抑制细胞凋亡的线粒体途径,这表明TGEV可能通过下调miR - 27b诱导细胞凋亡,并且miR - 27b可能成为治疗干预的靶点。