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 determine if miR-142-5p functions in PHEV infection, miR-142-5p antagomirs, also known as anti-miRs or blockmirs are a class of chemically engineered and modified oligonucleotides that prevent other molecules from binding to a desired site on an mRNA molecule, was injected into the cerebral cortex of infected mice with a stereotaxic apparatus to silence endogenous microRNA. The effectiveness and specificity of the antagomir in inhibiting endogenous miR-142-5p expression and enhancing Ulk1 expression has been demonstrated in mice ( Figure 9A) . Compared with only PHEV infection group, miR-142-5p antagomir treatment significantly induced ulk1 FIGURE 8 | Inhibition of Ulk1 blocks normal dendrites formation. Primary cortical neurons (14 DIV) were incubated with the designated constructs or PHEV. At 16 DIV, neurons were stained with antibodies against MAP2 (green) or PHEV (red). Representative images were taken with the same magnification. Compared to control primary cortical neurons, the PHEV-infected (A), Ulk1-RNAi-transfected (B), or miR-142-5p mimics-treated neurons (C) showed stunted spine length, complex arborization and heterogeneous spine size. But, Ad5-Ulk1 and miR-142-5p inhibitor treatments made less significance. Notably, numerous irregular membrane blebs in PHEV-infected neurons gathered a large number of virus particles (arrows). Data represent the means of n = 50 cells per condition counted in triplicate ± s.d. In each histogram, the y-axis represents the percentage of neurons, and the x-axis represents the number of spines at certain lengths (the middle panel) or the number of branches (the right panel).