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

The validation of microRNA-mRNA interactions was performed using the Molecular Probes' fluorescence-based Electrophoretic Mobility Shift Assay (EMSA) Kit (Invitrogen, Gaithersburg, MD) according to the manufacturer's protocol. For the binding assays, the following RNA and DNA oligonucleotides (Sigma-Aldrich, Madrid, Spain) were designed and used: miR-142, an RNA sequence corresponding to the mature form of miR-142-5p; Ulk1-UTR, a 23-mer RNA sequence for the 3 ′ UTR corresponding to Ulk1 with the target site for miR-142-5p; anti-miR-142, a modified antisense oligodeoxynucleotide complementary to the sequence of the FIGURE 1 | Validation of miR-142-5p expression in vivo and in vitro. (A) qRT-PCR analysis of miR-142-5p expression in the cerebral cortex of mice infected, or not infected, with PHEV. (B) qRT-PCR analysis of miR-142-5p expression in primary cortical neurons. miR-142-5p expression normalized to U6 was examined, and the data are presented as the means ± SEM (n = 6). (C) ISH analysis in frozen section showed miR-142-5p RNA expression (red) in the prefrontal cortex and hippocampus. (D) ISH analysis in primary cortical neurons showed miR-142-5p (red) was up-regulated by PHEV. (E) Quantitative analyses of miR-142-5p RNA in primary cortical neurons infected, or not infected, with PHEV. The y-axis is the average miR-142-5p intensity using arbitrary fluorescence unit (AFU), **P < 0.01. (F) Two-color immunofluorescence staining of miR-142-5p RNA probe (red) and anti-PHEV (green) in cortical neurons in mice. Partial green staining co-localized with red staining, and several dendrite protrusions appear to be yellow (Arrow). mature form of miR-142-5p; and anti-miR-142MIS, an antisense oligodeoxynucleotide containing 11 mismatches compared to anti-miR-142. All RNA and DNA oligonucleotides were purchased from Sigma-Aldrich (Madrid, Spain), and their specific sequences are listed in the Table S1. The corresponding RNA or DNA molecules were incubated in binding buffer (750 mM KCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 50 mM Tris, pH 7.4) for 30 min at 37 • C, and the reaction products were then separated on a 10% non-denaturing polyacrylamide gel. After staining the gel with SYBR R Green solution for 20 min in the dark, it was photographed using 300 nm UV transillumination.