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 confirm that miR-142-5p bound to the Ulk1 mRNA 3 ′ UTR, we performed a luciferase reporter assay, which involved the wild-type or mutant-type Ulk1 3 ′ UTR ( Figure 5A) . Transfection with miR-142-5p mimics in neurons specifically decreased the activity of a luciferase reporter gene fused to the wild-type Ulk1 3 ′ UTR, whereas overexpression of the unrelated miR-21a-5p microRNA had no significant effect on the expression of this reporter construct (Figure 5B, black bars) . The effect of miR-142-5p on translation of the luciferase mRNA was dependent on the presence of the miR-142-5p cognate binding site within the 3 ′ UTR, and expression of a luciferase reporter containing the mutant-type Ulk1 3'UTR was unaffected by the presence of exogenous miR-142-5p ( Figure 5B , white bars). In contrast, miR-142-5p inhibitors led to a statistically increased expression of wild-type Ulk1 3'UTR reporter ( Figure 5C , black bars), but less effect on the mutant one ( Figure 5C , white bars). Moreover, analysis of Ulk1 expression in lysates from miR-142-5p mimics transfected primary cortical neurons showed a dose-dependent decrease in the level of endogenous Ulk1 protein, whereas delivery of its inhibitor led to an increase in protein expression ( Figure 5D ). Further investigation determined that Ulk1 level was inhibited in vivo after the miR-142-5p antagomir was injected into the brains of mice with a stereotaxic apparatus (Figure 9A , left panel). Taken together, these data indicated that miR-142-5p inhibited Ulk1 expression by binding to a single site present in the Ulk1 mRNA 3 ′ UTR, and Ulk1 was a nerve injury associated mRNA down-regulated by miR-142-5p in PHE.