miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1

Li, Zi; Lan, Yungang; Zhao, Kui; Lv, Xiaoling; Ding, Ning; Lu, Huijun; Zhang, Jing; Yue, Huiqing; Shi, Junchao; Song, Deguang; Gao, Feng; He, Wenqi

To gain insight into the mechanisms by which miR-142-5p regulates axon morphology during PHEV infection, we sought to identify miR-142-5p target mRNAs. Potential targets of miR-142-5p were predicted using the microRNA target prediction databases, including TargetScan, miRBase, and and miRwalk. For this analysis, we focused on a set of 10 genes that we recently identified in a screen for mRNAs in which translation was reduced in neurons upon treatment with PHEV . Among these mRNAs, Ulk1 was of particular interest and was found to contain conserved 3'UTR sequence elements that were partially complementary to mouse miR-142-5p. Using an electrophoretic mobility shift assay (EMSA), we demonstrated that Ulk1 mRNA and miR-142-5p interacted in vitro ( Figure 4A ). qRT-PCR and Western blotting analysis showed that Ulk1 expression levels were all down-regulated reciprocally with miR-142-5p in the primary cortical neurons after PHEV treatment, and that Ulk1 mRNA expression was nearly 1.2-fold lower compared to the expression in normal neurons within 60 hpi ( Figure 4B) . Consistent results were observed in mice infected with PHEV, and Ulk1 expression was reduced to the lowest level in the cerebral cortex of infected mice at 5 dpi ( Figure 4C) . Moreover, an immunocytochemistry assay further confirmed that Ulk1 expression decreased in the injured brain tissue compared to the normal brain tissue ( Figure 4D) . Therefore, we hypothesized that Ulk1 mRNA was a putative miR-142-5p target gene and may be involved in PHEV-induced neuronal damage.