Cells, virus, and primers
In the present study, MDBK cells, obtained from the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Biological Industries, Beit Haemek, Israel) supplemented with 100 IU of penicillin and 100 mg of streptomycin per milliliter at 37℃ in an incubator with 5% CO2. IBRV was purchased from the China Institute of Veterinary Drug Control. Primers, mimics, and inhibitor of miRNAs were synthesized by Shanghai Sangon Biological Engineering Technology Company Limited (Shanghai, China). The sequences are shown in Additional file 11: Table S11.
Sample preparation
MDBK cells were seeded at a density of 3 × 106 cells per plate into nine 100 mm culture dishes.MDBK cells were divided into three experimental groups, i.e., Mock group with 3 duplications (Mock-1, Mock-2, and Mock-3), IBRV infection for 6 hour group (IBRV1) with 3 duplications (IBRV1-1, IBRV1-2, and IBRV1-3) and IBRV infection for 24 hour group (IBRV2) with 3 dupications (IBRV2-1, IBRV2-2, and IBRV2-3). The IBRV-infected groups were inoculated with 1.5 multiplicity of infection (MOI) virus when the cells reached confluence (85%). The same volume of serum-free DMEM was added to the Mock groups, and the cells from these groups were collected 6 hours and 24 hours later. Then, 1 mL of TRIzol (Invitrogen, Carlsbad, CA, USA) was added, and the cells were stored at -80°C after liquid nitrogen flash freezing.
High-throughput sequencing
High-throughput sequencing was performed by Gene Denovo Biotechnology Co. (Guangzhou, China). After total RNA was extracted using a TRIzol reagent kit (Invitrogen, Carlsbad, CA, USA), RNA molecules in a size range of 18–30 nt were enriched by polyacrylamide gel electrophoresis. Then, 3′ adapters were added, and 36–44 nt RNAs were enriched. Subsequently, 5′ adapters were ligated to the RNAs. The ligation products were reverse transcribed by polymerase chain reaction (PCR) amplification, and 140–160 bp size PCR products were enriched to generate a complementary DNA (cDNA) library. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads (Invitrogen, Carlsbad, CA, USA). The enriched mRNA was then fragmented into short fragments using fragmentation buffer and reverse transcribed into cDNA using the NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA). The purified double-stranded cDNA fragments were end repaired, a base added, and ligated to Illumina sequencing adapters. The ligation reaction was purified with AMPure XP Beads (1.0X) (Beckman Coulter, USA) and PCR amplified. The resulting cDNA library was sequenced using Illumina Novaseq 6000 (Guangzhou, China).
Alignment of miRNAs and mRNAs
The low-quality reads were removed to obtain clean tags, which were aligned with small RNAs in the GeneBank database and Rfam database to identify and remove ribosomal RNAs, small cytoplasmic RNAs, small nucleolar RNAs, small nuclear ribonucleic acids, and transfer RNA. The rest of the clean tags were then aligned with the ARS-UCD1.2 reference genome and searched against the miRBase database to identify existing miRNAs. Unmapped miRNAs were aligned with other species. All the unannotated tags were aligned with the ARS-UCD1.2 reference genome. According to the genome positions and hairpin structures predicted by miRDeep2 software (https://github.com/rajewsky-lab/mirdeep2), novel miRNA candidates were obtained. An index of the reference genome was built, and paired-end clean reads were mapped to the ARS-UCD1.2 reference genome using HISAT2 (v2.0.4).
Quantification of miRNA and mRNA abundance
The miRNA expression level was calculated and normalized to transcripts per million (TPM) using the following formula:
$$TPM=\frac{T{10}^{6}}{N}$$
where T is the actual miRNA count, and N is the total count of clean tags (existing, known, and novel miRNAs).
The mRNA expression level was calculated and normalized to fragment per kilobase of transcript per million mapped reads (FPKM) using the formula below:
$$FPKM=\frac{{10}^{6}C}{NL/{10}^{3}}$$
where RPKM is the expression level of a given gene A, C is the number of fragments that are uniquely aligned to gene A, N is the total fragment number that is aligned to the reference gene, and L is the number of bases of the coding region of gene A.
Prediction of miRNAs targets
RNAhybrid (v2.1.2) + svm_light (v6.01), Miranda (v3.3a), and TargetScan (v7.0) were used to predict targets of the miRNAs. The intersection of the target genes of the differentially expressed miRNAs and identified mRNAs was chosen as candidate targets of miRNAs.
GO and KEGG enrichment analysis of differentially expressed miRNAs and mRNAs
Functional annotation was performed using GO (http://www.geneontology.org/) enrichment analysis and KEGG (http://www.genome.ad.jp/kegg/) pathway analysis. We performed functional annotation of differentially expressed mRNAs and target genes of differentially expressed miRNAs to cellular component (CC), molecular function (MF), and biological process (BP) using the GO database. In the enrichment analysis, the number of genes in each GO term was counted. For the KEGG analysis, the top 25 entries in the KEGG pathways were exhibited.
miRNA-mitochondria-related target gene regulatory networks
The miRNA-mitochondria-related target gene regulatory networks were constructed using a combination of miRNA-mitochondria-related target gene pairs, and the regulatory networks were visualized using Cytoscape (v3.7.0) (http://www.cytoscape.org/).
Quantification of miRNAs by reverse transcription-quantitative PCR (RT-qPCR)
Ten miRNAs were chosen from differentially expressed miRNAs using a simple random sampling method generated using Microsoft Office Excel (Microsoft Office Excel 2016, Microsoft Corporation, Redmond, USA). TRIzol (Invitrogen, Carlsbad, CA, USA) was used to extract total RNA from the Mock and IBRV groups. RNA was reverse-transcribed using the Hifair® III 1st Strand cDNA Synthesis Kit (gDNA digester plus) (Yeasen, Shanghai, China), in accordance with the manufacturer’s protocol. cDNA was amplified by the 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using 2x RealStar Green Fast Mixture with ROX Ⅱ (GenStar, Beijing, China). All data were calculated using the 2−ΔΔCt method, and the miRNA level of each sample was normalized according to U6 expression. Each group comprised three duplicate wells.
Transfection of miRNAs
To confirm whether miR-10a and miR-182 played a role in mitochondrial damage, we investigated the biological functions of miR-10a and miR-182 in MDBK cells by overexpression and inhibition. The miRNA mimics and miRNA inhibitor were transfected into MDBK cells to overexpress or inhibit the expression of miR-10a and miR-182. The transfection efficiency of miR-10a and miR-182 was verified by RT-qPCR, and the biological functions of miR-10a and miR-182 were verified by flow cytometry. Mimics, inhibitors, and negative control oligonucleotides of miR-10a and miR-182 were purchased from Sangon Biotech Co., Ltd (Shanghai, China). MDBK cells were cultured to 60–70% confluence after being seeded onto six-well plates. Transfection of cells with oligonucleotides was performed using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, CA, USA) at a final concentration of 100 nM in line with the manufacturer’s instructions. Twelve hours later, the transfected cells were inoculated with 1.5 MOI IBRV. After 6 and 24 hours of inoculation, the cells were harvested for further study.
Determination of intracellular mPTP levels
The generation of mPTP was determined according to the manufacturer’s instructions using an mPTP assay kit (BestBio, Shanghai, China). Each sample was treated with 5 µl of BbcellProbe™ M61 probe (200 µM) for 15 minutes and quencher for 15 minutes at 37°C. Opening of mPTP results in a decrease of fluorescence. Fluorescence was measured by flow cytometry (Mindray, Shenzhen, China), with 10,000 events collecting.
Determination of intracellular MMP levels
The MMP was determined using a JC-1 Mitochondrial Membrane Potential assay kit (Beyotime, Jiangsu, China). MDBK cells were washed with serum-free DMEM and incubated in JC-I working solution for 20 minutes in the dark at 37°C. After washing, the cells were re-suspended with JC-1 dye buffer, and JC-1 monomer fluorescence distribution and j-aggregates were measured. The fluorescence was measured by flow cytometry(Mindray, Shenzhen, China), with 10,000 events collecting. Mitochondrial depolarization of MDBK cells in the Mock and IBRV groups were calculated by a decrease in the red/green fluorescence intensity ratio.
Statistical analysis
The data are presented as the means ± standard error of mean (SEM). Statistical comparisons were performed using an unpaired Student’s t-test. the statistical significance was evaluated by Graphpad Prism 8.0 software. Relative to the control, * p < 0.05 and ** p < 0.05versus the control were regarded as significant.