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Gene expression profiling of porcine skeletal muscle satellite cells after poly(I:C) stimulation
Gene 695 (2019) 113–121
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Research paper
Gene expression profiling of porcine skeletal muscle satellite cells after poly (I:C) stimulation
T
Thuy-Nhien Tran-Thia, Sheng Wanga, Adeyinka Abiola Adetulaa, Cheng Zoua, ⁎ Abdullah Ibne Omara,b, Jian-Lin Hanc,d, Ding-Xiao Zhanga, Shu-Hong Zhaoa, a
Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education, Key Laboratory of Pig Genetics and Breeding of the Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China b National Engineering Laboratory for Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China c CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, PR China d International Livestock Research Institute (ILRI), Nairobi 00100, Kenya
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine Satellite cell Poly(I:C) Differentially expressed gene Signaling pathway
Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48 hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the upregulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.
1. Introduction Previous investigations have revealed that satellite cells play a crucial role in the formation/regeneration, development and reparation of animal skeletal muscle (Shi and Garry, 2006; Relaix and Zammit, 2012). In addition, the chemotaxis, proliferation and differentiation of skeletal muscle satellite cells are regulated by immune, nervous and vascular systems as well as autocrine factors (Nierobisz and Mozdziak, 2008). During differentiation stage, satellite cells are shaped into multinucleated myotubes and contribute to the hypertrophy of animal
skeletal muscle (Briata et al., 2012). Under normal circumstances, satellite cells are quiescent in their niche between the sarcolemma and basal lamina of skeletal muscle fibers (Mauro, 1961). In the inflammatory response of muscle injury, these cells are immediately activated to proliferate for the repair and regeneration of new myoblasts (Boldrin et al., 2010). The proliferation of satellite cells are affected by many physiological and pathologic elements such as exercises, aging, poisonous agents, neurological damages, atrophies and myopathies via various signaling pathways (Nierobisz and Mozdziak, 2008). The proliferation of skeletal muscle satellite cells is regulated by canonical Wnt-
Abbreviations: PSCs, porcine satellite cells; DEGs, differentially expressed genes; Poly(I:C), Polyinosinic:polycytidylic acid; dsRNA, Double-stranded Ribonucleic acid; FPKM, fragments per kilobase of transcript per million mapped reads; FC, fold change; cDNA, DNA complementary to RNA; qPCR, quantitative real-time PCR; ANOVA, analysis of variance; PBS, Phosphate buffered saline; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; BPs, biological processes; CCs, cellular components; MFs, molecular functions; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ⁎ Corresponding author at: Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. E-mail addresses: [email protected] (T.-N. Tran-Thi), [email protected] (S. Wang), [email protected] (A.A. Adetula), [email protected] (C. Zou), [email protected] (A.I. Omar), [email protected] (J.-L. Han), [email protected] (D.-X. Zhang), [email protected] (S.-H. Zhao). https://doi.org/10.1016/j.gene.2018.12.059 Received 7 July 2018; Received in revised form 21 December 2018; Accepted 30 December 2018 Available online 08 January 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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paraformaldehyde for 15 min. Next, 0.3% Triton X-100 was added to PSCs for 10 min at room temperature. After that, blocking of unspecific binding of the antibody and permeabilization were performed using 3% bovine serum albumin, 0.3% Triton X-100 and 10% fetal bovine serum in PBS solution for 2 h at room temperature. PSCs were incubated with the primary anti-myosin (1:1000) at 4 °C overnight. The anti-mouse IgG was used as the secondary antibodies for 2 h at room temperature. Then, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by the same Olympus inverted microscope system.
signalling pathway via the overexpression of Wnt1, Wnt3a and Wnt5a proteins (Otto et al., 2008). The regulation between self-renewal and differentiation of satellite cells is directed by Fas-associated death domain (Cheng et al., 2014). Polyinosinic:polycytidylic acid (poly(I:C)), a synthetic double stranded RNA (dsRNA), has been applied to mimic viral infection to induce the immune response models in experimental animals. Poly(I:C) induced an antiviral response in many cell types such as human corneal epithelial cells (Kumar et al., 2006) and lung endothelial cells (Huang et al., 2016), prompted the apoptosis of human CD34+ cells (Liu et al., 2012), but inhibited the differentiation of mouse preadipocytes (Yu et al., 2016). Therefore, in this study, we used poly(I:C) to induce the response of porcine satellite cells (PSCs) to a mimicking viral infection, for a depth understanding of the response mechanisms of PSCs. RNA-Seq is one of the next-generation sequencing technologies and is widely used for transcription profiling because of its high sensitivity, reproducibility, dynamic range of expression and base pair resolution. This technology was used to study the transcriptomic profiles of PSCs under proliferation and differentiation phases (Jensen et al., 2017). Considering the importance of satellite cells for the regeneration and development of animal skeletal muscle, we executed this study to examine the effect of poly(I:C) stimulation on PSCs during the proliferation and differentiation phases. This study has the potential to reveal novel genes and pathways responsible for immune barrier to self-protection of poly(I:C)-induced PSCs.
2.4. RNA quality control, sequencing and analysis A total of 21 RNA samples were isolated from PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation using the TRIzol Reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA 6000 Nano assay of the Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., CA, USA). Twenty-one first strand cDNA libraries were synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). The Solexa sequencing was implemented on the Illumina Hiseq 4000 Systems (NEB, USA). The raw sequence data files discussed in this experiment have been deposited in NCBI's Gene Expression Omnibus (GEO) (Series GSE112527) (https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE112527) (accession numbers included untreated PSCs at 0 h: GSM3072241, GSM3072242 and GSM3072243; untreated PSCs at 12 h: GSM3072244, GSM3072245 and GSM3072246; treated PSCs at 12 h: GSM3072247, GSM3072248 and GSM3072249; untreated PSCs at 24 h: GSM3072250, GSM3072251 and GSM3072252; treated PSCs at 24 h: GSM3072253, GSM3072254 and GSM3072255; untreated PSCs at 48 h: GSM3072256, GSM3072257 and GSM3072258; treated PSCs at 48 h: GSM3072259, GSM3072260 and GSM3072261). The quality of obtained sequencing data was controlled using the FastQC software (Babraham Bioinformatics, Cambridge, UK) and trimmed using the Trimmomatic tool (Bolger et al., 2014). The adapters were trimed, all reads were mapped to and sequences were identified based on the Ensembl Sus scrofa 11.1.90 set GTF annotation genome (available at ftp://ftp.ensembl.org/pub/release-90/gtf/sus_scrofa/) using the HISAT2 program (Kim et al., 2015). Afterwards, the Stringtie assembler was used to estimate gene expression and transcript levels via the FPKM (fragments per kilobase of transcript per million mapped reads) approach (Pertea et al., 2015). Thirteen comparison libraries were performed using the data obtained from the RNA-Seq, including untreated groups: 0 h vs 12 h untreated, 12 h vs 24 h untreated, 24 h vs 48 h untreated, 0 h vs 24 h untreated and 0 h vs 48 h untreated; and treated groups: 0 h vs 12 h treated, 12 h vs 24 h treated, 24 h vs 48 h treated, 0 h vs 24 h treated and 24 h vs 48 h treated; and between treated and untreated groups: 12 h untreated vs treated, 24 h untreated vs treated and 48 h untreated vs treated groups. Then, the differentially expressed genes (DEGs) among different comparison libraries were identified with q-value < 0.05 and absolute of log2 fold change (|log2FC|) ≥ 1.5 using the Ballgown package (Frazee et al., 2015).
2. Materials and methods 2.1. Porcine satellite cells and poly(I:C) stimulation All experimental procedures were strictly conducted following the guidelines established by the Standing Committee of Hubei People's Congress (No. 5) and approved by the Scientific Ethics Committee of Huazhong Agricultural University. PSCs were isolated from five-day -old piglet hind-limb muscles. The isolated PSCs were placed in 6-well plates and incubated in the growth media (Bovine serum, DMEM, Gentamycin and AA solution) at 37 °C and 5% CO2 until their confluence around 24 hours (h) of culture. Then, PSCs were treated with poly(I:C) at 10 μg/mL for 0, 12, 24, and 48 h while the untreated PSCs were included as negative controls. Next, PSCs were rinsed with phosphate-buffered saline (PBS) solution and induced to differentiate by the differentiation media containing 5% horse serum. The triplicate samples at 0, 12, 24 and 48 h of poly(I:C) stimulation were observed under an Olympus inverted microscope system (Olympus, Tokyo, Japan) (Fig. 2(A-H)) and then harvested using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for further analyses. 2.2. Proliferation assay Cell viability was analyzed using 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) assay kit according to the manufacturer's instructions (Sigma Chemical Co.). Briefly, PSCs were cultured with 10 μg/mL poly(I:C) in a 96-well plate at a density of 2 × 104 cells per well. After 12 h of poly(I:C) stimulation, PSCs were incubated at 37 °C with 10 μL of MTT (5 mg/mL) solution for 4 h. Then, the medium was removed, and 100 μL of Formazan solvent was added into each well for 1 h at 37 °C. The absorbance at 570 nm was measured using an EnSpire Multimode Plate Reader (PerkinElmer). The percentage of the absorbance value was used to evaluate the cell viability.
2.5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses The up- and down-regulated genes were separated to investigate the functions of genes and pathways, using the version of Sus scrofa annotation in Database for Annotation, Visualization, and Integrated Discovery platform (DAVID, https://david.ncifcrf.gov/). The GO terms (including biological processes (BPs), cellular components (CCs), and molecular functions (MFs)) were utilized to detect the functions of genes. The pathway detection was identified by the KEGG search pathway tools (Kanehisa and Goto, 2000).
2.3. Differentiation assay The differentiation of PSCs was observed by myosin immunofluorescence staining. Briefly, PSCs after proliferation were induced to differentiate in the differentiation media containing 5% horse serum. Then, PSCs were rinsed twice with PBS solution and fixed with 4% 114
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observations of PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation are illustrated in Fig. 2(A–H).
2.6. Quantitative RT-PCR (qPCR) and statistical analysis To investigate the levels of gene expressions after poly(I:C) stimulation, total RNA obtained from the samples was converted into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Bio Inc., Otsu, Japan). The qPCR was performed using the SYBR® Green Real-time PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan) on the CFX384 Touch™ Real-Time PCR Detection System following the manufacturer's instructions (Bio-Rad). The Oligo7 Primer Analysis software (Molecular Biology Insights, Inc., Cascade, CO, USA) was applied to design and evaluate the primers for gene validations. The specific primer sequences used for qPCR are shown in Supplementary file 1. The qPCR was performed as follows: 1 cycle at 95 °C for 2 min, 40 cycles at 95 °C for 30 s, 60 °C for 20 s and 72 °C for 15 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize the expression levels. SPSS 21.0 software was utilized to estimate qPCR datasets with one-way analysis of variance (ANOVA) test (Ganger et al., 2017). The significant differences were considered at p-value < 0.05.
3.2. RNA-Seq and differentially expressed genes (DEGs) A total of 13 comparison libraries of PSCs were analyzed from the data obtained by the RNA-Seq method, including untreated and treated PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation. The summary of the RNA-Seq data from PSCs are shown in Supplementary file 2. Interestingly, we found 406 DEGs from 4 out of the 13 comparison libraries (q-value < 0.05 and |log2fold-change| ≥ 1.5), including 88 DEGs in 0 h vs 12 h treated, 119 DEGs in 12 h vs 24 h treated, 104 DEGs in 0 h vs 24 h treated and 95 DEGs in 24 h vs 48 h untreated comparison libraries. The numbers and expression levels of DEGs identified in each pair of the comparison libraries (0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated) are shown in Fig. 3(A–C) and Supplementary file 3, among which 241 were upregulated but 165 were down-regulated. The highest level of an upregulated gene was 8.26-fold change (FC) in 12 h vs 24 h treated comparison, whereas the lowest level of a down-regulated gene was −6.48 FC in 0 h vs 12 h treated comparison (Fig. 3(C)). Venn diagrams were used to distinguish the overlapped and unique DEGs among upand down-regulated genes. Among up-regulated genes, 71 DEGs were unique in 0 h vs 12 h treated comparison libraries, whereas 15 DEGs were overlapped between 12 h vs 24 h treated and 0 h vs 24 h treated comparison libraries (Fig. 3(A)). Similarly, 20 down-regulated genes were overlapped in 12 h vs 24 h treated and in 0 h vs 24 h treated comparison libraries (Fig. 3(B) and Supplementary file 4). The hierarchically clustered heatmaps were constructed to classify the transcriptional profiles of the 406 DEGs among the comparison libraries of untreated and treated PSC groups (Fig. 4 and Fig. 5(A-D)). The overall heatmap for all samples illustrated the differences (qvalue < 0.05) between the groups and among poly(I:C) stimulations during the proliferation and differentiation phases (Fig. 4). During the proliferation phase, there was no significant difference (q-value ≥ 0.05) between the untreated groups at 0 h and 12 h of the stimulation, whereas there were significant differences (q-value < 0.05) between treated groups at 0 h and 12 h of the stimulation. Among the overall expression, four comparison libraries were identified with significant DEGs (Fig. 5(A-D)). The hierarchically clustered heatmaps showed
3. Results 3.1. Proliferation and differentiation phases of PSCs To study the gene expression levels at pre- and post-poly(I:C) stimulation during the proliferation and differentiation phases, we isolated and cultured PSCs from the piglet hind limb muscles. The cell visibility of PSCs during the proliferation phase was measured by MTT assay (Fig. 1(A)). The results showed that there was no significant difference in cell visibility between untreated (100%) and treated (94.43%) groups, suggesting that mimicking viral infection did not induce the death of PSCs during the proliferation phase. However, the cell fusion and myotube formation during the differentiation phase of PSCs were definitely distinguishable between untreated and treated groups when they were observed using the myosin immunofluorescence staining (Fig. 1(B)). During the differentiation stage, the cell fusion and myotubes are constructed by an arrangement of adjacent cells. The myosin immunofluorescence staining results indicated that the elongated shape of myotubes in the untreated groups was more visibly distinct than that in the treated groups (Fig. 1(B)). The microscope
Fig. 1. PSCs after poly(I:C) stimulation. (A) Cell viability after poly(I:C) stimulation during the proliferation phase. (B) Myosin immunofluorescence staining of PSCs during the differentiation phase. Nucleus was stained with DAPI (blue). Scale bars: 100 μm. Magnification: 100×. 115
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Fig. 2. Phase microscopy images of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation. Proliferation phase was performed within 12 h while differentiation phase was performed between 24 and 48 h of poly(I:C) stimulation. (A), (B), (C) and (D) are the untreated PSCs at 0, 12, 24 and 48 h of the stimulation, respectively. (E), (F), (G) and (H) are the treated PSCs at 0, 12, 24 and 48 h of the stimulation, respectively.
were associated with muscle contraction, muscle structure development, actin filament-based process and cytoskeleton regulation, while down-regulated genes were related to viral genome replication, immune response and response to external stimulus (Fig. 6(C-D)). In 0 h vs 24 h treated comparison libraries, up-regulated genes were enriched in BPs of the regulations of defense response, regulation of response to stress, regulations of cell communication and signal transduction (Fig. 6(E)); whereas cell cycle process, regulation cell cycle and mitotic cell cycle phase transition were represented among down-regulated genes (Fig. 6(F)). The DEGs involved in CCs and MFs were less than in BPs; in which, most up-regulated genes during the differentiation phase were engaged in the development of muscle, whereas down-regulated genes belonged to cellular processes (Supplementary file 5). The DEGs were mapped to canonical pathways based on KEGG database (Supplementary file 6 and Table 1). Interestingly, the significant pathways emerged in 0 h vs 12 h treated comparison libraries included Influenza A, proteasome, Hepatitis B and C, measle, and Herpes simplex infection pathways, RIG-I-like receptor, TLR and chemokine signaling pathways, and cytosolic DNA-sensing pathway (Table 1). The pathways related to the cell cycle and DNA replication in
significant differences (q-value < 0.05) in comparisons of 0 h vs 12 h treated libraries, 12 h vs 24 h treated libraries, 0 h vs 24 h treated libraries and 24 h vs 48 h untreated libraries (Fig. 5(A-D)). Therefore, these results distinguished the changes in gene expression levels of PSCs after poly(I:C) stimulation during the proliferation and differentiation phases. 3.3. GO and KEGG pathway analyses Functional classifications based on GO categories annotated the DEGs from comparison libraries. The GO term results are shown in Fig. 6(A-H) and Supplementary file 5. The representative collections of BPs were selected and depicted by both the level of enrichments and the number of genes. During the proliferation phase, up-regulated genes found in the BPs of 0 h vs 12 h treated comparison libraries were related to immune system such as regulation of innate immune response, immune effector processes and defense response to virus; whereas downregulated genes were associated with muscle organ development, muscle structure development and response to growth factor (Fig. 6(AB)). In 12 h vs 24 h treated comparison libraries, up-regulated genes
Fig. 3. The numbers and expression levels of DEGs among comparison libraries. Venn diagrams present the numbers overlapped of DEGs in comparison libraries (0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated groups). (A) and (B) Distribution of up- and down-regulated genes between the comparison libraries, respectively. (C) The average expression levels of DEGs between comparison libraries with Mean ± SEM. Error bars represent standard error (SEM) of the mean. 116
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Fig. 4. The overall heatmap of DEGs between the untreated and treated PSCs during the proliferation and differentiation phases. Each row represents a sample while each column represents a gene. The color key value shows the minimum to maximum (0 to 10) expression of genes by binary logarithms of (FPKM + 1).
(IFIH1), interferon-stimulated gene 15 (ISG15) and C-C motif chemokine ligand 5 (CCL5) related to immune response were highly expressed (p-value < 0.05) at 12 h of the stimulation, whereas their expression levels decreased between 24 and 48 h of the stimulation during the differentiation phase (p-value < 0.05). Besides, three genes including myosin heavy chain 4 (MYH4), leiomodin-3 (LMOD3) and tripartite motif containing 72 (TRIM72) related to muscle differentiation were also highly expressed (p-value < 0.05) during the differentiation phase, however, their expression levels were lower in treated than untreated groups. The validation results are shown in Fig. 7.
the comparison libraries of 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated groups are shown in Supplementary file 6. 3.4. Validation analysis We selected nine DEGs which were involved in signaling pathways and BPs in 0 h vs 12 h treated comparison libraries, to validate their expression levels uncovered from the RNA-Seq analysis. Six up-regulated genes including DExD/H-Box helicase 58 (DDX58), radical S adenosyl methionine domain containing 2 (RSAD2), 2′-5′-oligoadenylate synthetase 2 (OAS2), interferon induced with helicase C domain 1
Fig. 5. The DEGs of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation (A), (B), (C), (D) for the DEGs in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. Each column represents a sample while each row represents a gene. The color key value shows the minimum to maximum (0 to 10) expression of genes by binary logarithms of (FPKM + 1). 117
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Fig. 6. The distinctive BPs of DEGs among comparison libraries. (A) and (B) for BPs of up- and down-regulations in 0 h vs 12 h treated comparison libraries, respectively; (C) and (D) for BPs of up- and down-regulations in 12 h vs 24 h treated comparison libraries, respectively; (E) and (F) for BPs of up- and downregulations in 0 h vs 24 h treated comparison libraries, respectively; (G) and (H) for BPs of up- and down-regulations in 24 h vs 48 h untreated comparison libraries, respectively. (UN: untreated; TR: treated; up: up-regulation; down: down-regulation).
4. Discussion
regulated genes were associated with protein impairment, growth and differentiation of PSCs (Jensen et al., 2012). However, the gene expression levels during viral infection of skeletal muscle satellite cells have not been unconcealed. In this study, we stimulated PSCs using poly(I:C) to induce immune responses and then performed the RNA-Seq analysis to determine DEGs and the signaling pathways of PSCs undergoing mimicking viral infection. Our results showed that after poly(I:C) stimulation, the clusters of up-regulated genes were associated with BPs related to defense and immune responses, including type I interferon (IFN-I) production (Supplementary file 5). Previous studies indicated that the mimicking
PSCs are not only known for their essential role in muscle formation and development, but also their ability of excellent self-renewal after muscle injury. The transcriptome profiling of skeletal muscle satellite cells during myotube formation revealed that the up-regulated genes were involved in the muscle contraction and muscle system process in several species such as pig, caprine and bovine (Tripathi et al., 2014; Tong et al., 2015; Jensen et al., 2017). In human, the skeletal muscle satellite cells were identified as the infectious target of Chikungunya virus (Ozden et al., 2007). In the acute physical stimulation, most up118
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Table 1 The signaling pathways identified in 0 h vs 12 h treated comparison libraries. Pathways
Terms
Gene count
p-value
Genes
Enrichment
ssc05164 ssc05168 ssc04622 ssc04623 ssc04620 ssc05160 ssc05162 ssc05161 ssc04062 ssc03050
Influenza A Herpes simplex infection RIG-I-like receptor signaling pathway Cytosolic DNA-sensing pathway Toll-like receptor signaling pathway Hepatitis C Measles Hepatitis B Chemokine signaling pathway Proteasome
9 8 6 5 5 5 5 4 4 3
1.78E−07 5.19E−06 6.04E−06 8.99E−05 6.30E−04 0.001486 0.001714 0.021484 0.030565 7.18E−04
DDX58, IFIH1, IRF7, RSAD2, OAS2, CCL5, STAT1, FURIN, CXCL10 DDX58, IFIH1, IFIT1, IRF7, TAP1, OAS2, CCL5, STAT1 DDX58, IFIH1, ISG15, IRF7, DHX58, CXCL10 DDX58, MB21D1, IRF7, CCL5, CXCL10 IRF7, CXCL9, CCL5, STAT1, CXCL10 DDX58, IFIT1, IRF7, OAS2, STAT1 DDX58, IFIH1, IRF7, OAS2, STAT1 DDX58, IFIH1, IRF7, STAT1 CXCL9, CCL5, STAT1, CXCL10 PSME1, PSMB8, PSMB9
13.06 10.66 21.39 19.87 12.00 9.54 9.18 6.42 5.60 21.55
trigged by mediated TLR3 signaling pathway (Ning et al., 2011). In this study, transcriptional factor of IRF7 gene participated in TLR signaling pathway, suggesting its significant role in dictating the outcome of innate immune responses. The expressed genes associated with TLR signaling pathway were also related to chemokine and transcription factor families (also see Medzhitov, 2001) (Table 1). Chemokines play an essential role in inflammatory responses, immune modulators and lymphopoiesis (Zlotnik and Yoshie, 2000). An early investigation demonstrated that dsRNA induced the secretion of proinflammatory chemokines such as CCL5 in the bronchial epithelial cells (Gern et al., 2003). In our results, several chemokine members such as CCL5, C-X-C motif chemokine ligand 9 (CXCL9) and C-X-C motif chemokine ligand 10 (CXCL10) were up-regulated during poly(I:C) stimulation and involved in chemokine signaling pathway. Thus, our finding proposed that mimicking viral poly(I:C) stimulation activated the expression of immune and inflammatory response genes through TLR and chemokine signaling pathways in PSCs. These pathways are representatives which were induced by poly(I:C) stimulation in many cell types (Gern et al., 2003; Yan et al., 2013; Yu et al., 2016). As mention above, the expressions of MYH4, LMOD3 and TRIM72 genes in treated group were lower than in the negative controls during the differentiation phase (24 to 48 h of the stimulation) (Fig. 7). The expression levels of marker genes such as LMOD3, myomesin 3 (MYOM3) and neurogenic locus notch homolog protein 3 (NOTCH3) associated with the muscle differentiation and self-renewal of muscle satellite cells were revealed in our study (Supplementary file 3), consistent with a recent study (Flamini et al., 2018). Previous studies demonstrated that MYH4 expression was high in fast skeletal muscle of pig (Brown et al., 2014). Besides, LMOD3 expression was induced in early differentiation period of skeletal muscle along with its strong actin filament (Yuen et al., 2014). In addition, the expression of TRIM72 gene also plays an important role in the differentiation period of skeletal muscle (Cai et al., 2009). All these lines of evidence indicated that the expression levels of MYH4, LMOD3 and TRIM72 genes in the myogenesis of skeletal muscle are crucial. Ultimately, we speculated that viral infection may affect the regulation of genes related to the regeneration and development of PSCs.
viral poly(I:C) stimulation induced innate immune response in adipocytes and suggested the indirect inhibition of preadipocyte differentiation through downstream signaling pathway (Yu et al., 2016). In our results, the expression levels of genes such as MYH4, LMOD3 and TRIM72 (Fig. 7) related to the differentiation of PSCs were reduced after poly(I:C) stimulation. Thus, our finding suggested that the mimicking virus may also inhibit the differentiation of PSCs, similar to the results of poly(I:C) treated adipocytes (Yu et al., 2016). After trauma injury, the expression of genes related to immune response, chemokine and inflammation were up-regulated in the early period, while the genes associated with proliferation and differentiation regulations were exhibited in the middle period (Aguilar et al., 2016). Similarly, we observed high expressions of DEGs related to immune response and viral processes during mimicking viral infection of PSCs in the early period. The changes in gene expression levels of PSCs after poly(I:C) stimulation were investigated via functional pathway analysis. We focused on the changes in 0 h vs 12 h treated comparison libraries because of remarkable signaling pathways related to immunity functions (Table 1 and Supplementary file 6). Poly(I:C) is recognized by the pattern recognition receptors of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) (Yu and Levine, 2011). In the airway epithelial cells, poly (I:C) induced the up-regulation of IFN-I gene through stimulation of the RIG-I/MAVS pathway (Dauletbaev et al., 2015). Besides, poly(I:C) also stimulated the expression of antiviral effector gene 2′,5′-oligoadenylate synthetase (OAS) and IFN-I gene in human corneal epithelial cells (Kumar et al., 2006). In our results, the expressions of genes related to IFN-I, proinflammatory cytokines and chemokines such as DDX58, IFIH1 and CCL5 were increased (Table 1 and Fig. 7). Interestingly, these DEGs participated in influenza type A pathway, RIG-I-like receptor and TLR signaling pathways, and Hepatitis B and C pathways (Table 1). DDX58, also known as RIG-I, played a crucial role in the regulation of dsRNA signal induction and activated the expression of IFN-I gene (Yoneyama et al., 2004). Also, RIG-I expression was induced by a variety of viruses such as porcine reproductive and respiratory syndrome (Zhang et al., 2000), influenza, measles, Ebola, vesicular stomatitis and hepatitis C (Loo and Gale Jr., 2011). The activation of RIG-I and IFN-I genes were stimulated via a nucleotide component in panhandle promoter region of influenza A virus (Liu et al., 2015). In the present study, mimicking viral poly(I:C) stimulation induced RIG-I expression in most signaling pathways such as influenza A, herpes simplex infection, RIG-I-like receptor, cytosolic DNA-sensing, measles, hepatitis B and C, reflecting a significant function of RIG-I in immune regulation. Additionally, TLR and chemokine signaling pathways were activated by poly(I:C) stimulation in our study. TLRs play important roles in the recognition of pathogen-associated molecular patterns in innate immune system (Kawasaki and Kawai, 2014). Of which, TLR3 recognizes dsRNA and its analog (e.g. poly(I:C)) (Lester and Li, 2014). The activities of several interferon regulatory factors (IRFs) were regulated by TLR signaling pathways. Specifically the activation of IRF7 gene was
5. Conclusions In summary, we performed an RNA-Seq analysis to investigate the DEGs of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation. We identified a number of DEGs related to immune system, regeneration and development of muscle. Influenza A, TLR and chemokine signaling pathways were identified in the poly(I:C) stimulation groups when compared to the untreated groups. Consequently, it is suggested that genes detected in this study are candidates for further analysis of regulatory mechanism of mimicking viral induced PSCs. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.12.059. 119
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Fig. 7. Validations of DEGs by qPCR analysis between untreated and treated PSCs. Blue bars represent the untreated PSCs while red bars represent the treated PSCs at 0, 12, 24 and 48 h of the stimulation. GAPDH is used as a housekeeping gene for normalizing their expression levels. Six genes (DDX58, RSAD2, OAS2, IFIH1, ISG15 and CCL5) were highly expressed in the treated PSCs at 12 h of the stimulation while three genes (MYH4, LMOD3 and TRIM72) were highly expressed in the untreated PSCs when compared to the treated PSCs during the differentiation phase (between 24 and 48 h). * Significant level at p-value < 0.05 is based on Fishers least significant difference post hoc test of one-way ANOVA analysis. Error bars represent standard error (SEM) of the mean.
Author contributions
China (CGIAR 31361140365 and 31372302), the National High Technology Plan of China (2013AA102502) and the Fund of Modern Industrial Technology System of Pig (CARS-35).
SHZ, JLH, SW and TNTT designed the experiments; TNTT and SW cultured and stimulated PSCs by poly(I:C); AIO and TNTT implemented qPCR experiments; DXZ, AAA and CZ analyzed data; TNTT wrote the manuscript; SHZ, JLH, AAA and TNTT revised manuscript. All authors read and approved the final manuscript.
Acknowledgements We would like to acknowledge Professor Xinyun Li for his precious advice and encouragement during the implementation of the experiment.
Funding This work was supported by National Natural Science Foundation of 120
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Conflicts of interest
synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34(+) cells. Exp. Hematol. 40, 330–341. Liu, G., Park, H.-S., Pyo, H.-M., Liu, Q., Zhou, Y., 2015. Influenza A virus panhandle structure is directly involved in RIG-I activation and interferon induction. J. Virol. 89, 6067–6079. Loo, Y.M., Gale Jr., M., 2011. Immune signaling by RIG-I-like receptors. Immunity 34, 680–692. Mauro, A., 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493–495. Medzhitov, R., 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145. Nierobisz, L.S., Mozdziak, P.E., 2008. Factors influencing satellite cell activity during skeletal muscle development in avian and mammalian species. Asian Australas. J. Anim. Sci. 21, 456–464. Ning, S., Pagano, J.S., Barber, G.N., 2011. IRF7: Activation, regulation, modification and function. Genes Immun. 12, 399–414. Otto, A., Schmidt, C., Luke, G., Allen, S., Valasek, P., Muntoni, F., Lawrence-Watt, D., Patel, K., 2008. Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J. Cell Sci. 121, 2939–2950. Ozden, S., Huerre, M., Riviere, J.P., Coffey, L.L., Afonso, P.V., Mouly, V., de Monredon, J., Roger, J.C., El Amrani, M., Yvin, J.L., Jaffar, M.C., Frenkiel, M.P., Sourisseau, M., Schwartz, O., Butler-Browne, G., Despres, P., Gessain, A., Ceccaldi, P.E., 2007. Human muscle satellite cells as targets of Chikungunya virus infection. PLoS One 2, e527. Pertea, M., Pertea, G.M., Antonescu, C.M., Chang, T.-C., Mendell, J.T., Salzberg, S.L., 2015. Stringtie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295. Relaix, F., Zammit, P.S., 2012. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development 139, 2845–2856. Shi, X., Garry, D.J., 2006. Muscle stem cells in development, regeneration, and disease. Genes Dev. 20, 1692–1708. Tong, H.L., Yin, H.Y., Zhang, W.W., Hu, Q., Li, S.F., Yan, Y.Q., Li, G.P., 2015. Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing. Cell. Mol. Biol. Lett. 20, 351–373. Tripathi, A.K., Patel, A.K., Shah, R.K., Patel, A.B., Shah, T.M., Bhatt, V.D., Joshi, C.G., 2014. Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq. Mech. Dev. 132, 79–92. Yan, K., Zhu, W., Yu, L., Li, N., Zhang, X., Liu, P., Chen, Q., Chen, Y., Han, D., 2013. Tolllike receptor 3 and RIG-I-like receptor activation induces innate antiviral responses in mouse ovarian granulosa cells. Mol. Cell. Endocrinol. 372, 73–85. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., Fujita, T., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737. Yu, M., Levine, S.J., 2011. Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: Key modulators of innate immune responses to double-stranded RNA viruses. Cytokine Growth Factor Rev. 22, 63–72. Yu, L., Liu, G., Yang, C., Song, X., Wang, H., 2016. Polyinosinic-polycytidylic acid inhibits the differentiation of mouse preadipocytes through pattern recognition receptormediated secretion of tumor necrosis factor-alpha. Immunol. Cell Biol. 94, 875–885. Yuen, M., Sandaradura, S.A., Dowling, J.J., Kostyukova, A.S., Moroz, N., Quinlan, K.G., Lehtokari, V.L., Ravenscroft, G., Todd, E.J., Ceyhan-Birsoy, O., Gokhin, D.S., Maluenda, J., Lek, M., Nolent, F., Pappas, C.T., Novak, S.M., D'Amico, A., Malfatti, E., Thomas, B.P., Gabriel, S.B., Gupta, N., Daly, M.J., Ilkovski, B., Houweling, P.J., Davidson, A.E., Swanson, L.C., Brownstein, C.A., Gupta, V.A., Medne, L., Shannon, P., Martin, N., Bick, D.P., Flisberg, A., Holmberg, E., Van den Bergh, P., Lapunzina, P., Waddell, L.B., Sloboda, D.D., Bertini, E., Chitayat, D., Telfer, W.R., Laquerriere, A., Gregorio, C.C., Ottenheijm, C.A., Bonnemann, C.G., Pelin, K., Beggs, A.H., Hayashi, Y.K., Romero, N.B., Laing, N.G., Nishino, I., Wallgren-Pettersson, C., Melki, J., Fowler, V.M., MacArthur, D.G., North, K.N., Clarke, N.F., 2014. Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. J. Clin. Invest. 124, 4693–4708. Zhang, X., Wang, C., Schook, L.B., Hawken, R.J., Rutherford, M.S., 2000. An RNA helicase, RHIV-1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13. Microb. Pathog. 28, 267–278. Zlotnik, A., Yoshie, O., 2000. Chemokines: A new classification system and their role in immunity. Immunity 12, 121–127.
The authors declare that they have no conflict of interest. References Aguilar, Carlos A., Pop, R., Shcherbina, A., Watts, A., Matheny Jr., Ronald W., Cacchiarelli, D., Han, Woojin M., Shin, E., Nakhai, Shadi A., Jang, Young C., Carrigan, Christopher T., Gifford, Casey A., Kottke, Melissa A., Cesana, M., Lee, J., Urso, Maria L., Meissner, A., 2016. Transcriptional and chromatin dynamics of muscle regeneration after severe trauma. Stem Cell Rep. 7, 983–997. Boldrin, L., Muntoni, F., Morgan, J.E., 2010. Are human and mouse satellite cells really the same? J. Histochem. Cytochem. 58, 941–955. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Briata, P., Lin, W.J., Giovarelli, M., Pasero, M., Chou, C.F., Trabucchi, M., Rosenfeld, M.G., Chen, C.Y., Gherzi, R., 2012. PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis. Cell Death Differ. 19, 478–487. Brown, D.M., Brameld, J.M., Parr, T., 2014. Expression of the myosin heavy chain IIB gene in porcine skeletal muscle: The role of the CArG-Box promoter response element. PLoS One 9, e114365. Cai, C., Masumiya, H., Weisleder, N., Pan, Z., Nishi, M., Komazaki, S., Takeshima, H., Ma, J., 2009. MG53 regulates membrane budding and exocytosis in muscle cells. J. Biol. Chem. 284, 3314–3322. Cheng, W., Wang, L., Yang, B., Zhang, R., Yao, C., He, L., Liu, Z., Du, P., Hammache, K., Wen, J., Li, H., Xu, Q., Hua, Z., 2014. Self-renewal and differentiation of muscle satellite cells are regulated by the Fas-associated death domain. J. Biol. Chem. 289, 5040–5050. Dauletbaev, N., Cammisano, M., Herscovitch, K., Lands, L.C., 2015. Stimulation of the RIG-I/MAVS pathway by Polyinosinic:Polycytidylic acid upregulates IFN-β in airway epithelial cells with minimal costimulation of IL-8. J. Immunol. 195, 2829–2841. Flamini, V., Ghadiali, R.S., Antczak, P., Rothwell, A., Turnbull, J.E., Pisconti, A., 2018. The satellite cell niche regulates the balance between myoblast differentiation and self-renewal via p53. Stem Cell Rep. 10, 970–983. Frazee, A.C., Pertea, G., Jaffe, A.E., Langmead, B., Salzberg, S.L., Leek, J.T., 2015. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat. Biotechnol. 13, 243–246. Ganger, M.T., Dietz, G.D., Ewing, S.J., 2017. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments. BMC Bioinf. 18, 534. Gern, J.E., French, D.A., Grindle, K.A., Brockman-Schneider, R.A., Konno, S., Busse, W.W., 2003. Double-stranded RNA induces the synthesis of specific chemokines by bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 28, 731–737. Huang, L.Y., Stuart, C., Takeda, K., D'Agnillo, F., Golding, B., 2016. Poly(I:C) induces human lung endothelial barrier dysfunction by disrupting tight junction expression of claudin-5. PLoS One 11, e0160875. Jensen, J.H., Conley, L.N., Hedegaard, J., Nielsen, M., Young, J.F., Oksbjerg, N., Hornshoj, H., Bendixen, C., Thomsen, B., 2012. Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity. Exp. Physiol. 97, 833–848. Jensen, J.H., Madsen, L.B., Panitz, F., Hornshøj, H., Nielsen, R.O., Bendixen, C., Oksbjerg, N., Thomsen, B., 2017. Transcriptome dynamics during proliferation and differentiation of porcine primary satellite cells. Gene Rep. 7, 169–175. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30. Kawasaki, T., Kawai, T., 2014. Toll-like receptor signaling pathways. Front. Immunol. 5, 461. Kim, D., Langmead, B., Salzberg, S.L., 2015. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360. Kumar, A., Zhang, J., Yu, F.S., 2006. Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology 117, 11–21. Lester, S.N., Li, K., 2014. Toll-like receptors in antiviral innate immunity. J. Mol. Biol. 426, 1246–1264. Liu, J., Guo, Y.M., Hirokawa, M., Iwamoto, K., Ubukawa, K., Michishita, Y., Fujishima, N., Tagawa, H., Takahashi, N., Xiao, W., Yamashita, J., Ohteki, T., Sawada, K., 2012. A
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Research paper
Gene expression profiling of porcine skeletal muscle satellite cells after poly (I:C) stimulation
T
Thuy-Nhien Tran-Thia, Sheng Wanga, Adeyinka Abiola Adetulaa, Cheng Zoua, ⁎ Abdullah Ibne Omara,b, Jian-Lin Hanc,d, Ding-Xiao Zhanga, Shu-Hong Zhaoa, a
Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction of the Ministry of Education, Key Laboratory of Pig Genetics and Breeding of the Ministry of Agriculture and Rural Affairs, Huazhong Agricultural University, Wuhan 430070, PR China b National Engineering Laboratory for Animal Breeding, Key Laboratory of Agricultural Animal Genetics, Breeding and Reproduction, Ministry of Agriculture and Rural Affairs, College of Animal Science and Technology, China Agricultural University, Beijing 100193, PR China c CAAS-ILRI Joint Laboratory on Livestock and Forage Genetic Resources, Institute of Animal Science, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100193, PR China d International Livestock Research Institute (ILRI), Nairobi 00100, Kenya
A R T I C LE I N FO
A B S T R A C T
Keywords: Porcine Satellite cell Poly(I:C) Differentially expressed gene Signaling pathway
Porcine satellite cells (PSCs) play a vital role in the construction, development and self-renewal of skeletal muscle. In this study, PSCs were exposed to poly(I:C) stimulation to mimic viral infection during the proliferation and differentiation phases at 0, 12, 24 and 48 hours (h) of the stimulation. The untreated and treated PSCs were analyzed by the RNA-Seq technology. There were 88, 119, 104 and 95 genes being differentially expressed in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. The GO terms analysis results showed that during the proliferation phase of treated PSCs, the upregulated genes related to the immune system were highly expressed. In addition, the gene expressions associated with muscle structure development in response to growth factor emerged during the differentiation phase of untreated PSCs. The biological pathways associated with Influenza A, Toll-like receptor and chemokine signaling were revealed in PSCs following poly(I:C) stimulation. The differentially expressed genes were confirmed by quantitative real-time PCR. These findings expanded our understanding of gene expressions and signaling pathways about the infiltrated mechanism of the virus into PSCs.
1. Introduction Previous investigations have revealed that satellite cells play a crucial role in the formation/regeneration, development and reparation of animal skeletal muscle (Shi and Garry, 2006; Relaix and Zammit, 2012). In addition, the chemotaxis, proliferation and differentiation of skeletal muscle satellite cells are regulated by immune, nervous and vascular systems as well as autocrine factors (Nierobisz and Mozdziak, 2008). During differentiation stage, satellite cells are shaped into multinucleated myotubes and contribute to the hypertrophy of animal
skeletal muscle (Briata et al., 2012). Under normal circumstances, satellite cells are quiescent in their niche between the sarcolemma and basal lamina of skeletal muscle fibers (Mauro, 1961). In the inflammatory response of muscle injury, these cells are immediately activated to proliferate for the repair and regeneration of new myoblasts (Boldrin et al., 2010). The proliferation of satellite cells are affected by many physiological and pathologic elements such as exercises, aging, poisonous agents, neurological damages, atrophies and myopathies via various signaling pathways (Nierobisz and Mozdziak, 2008). The proliferation of skeletal muscle satellite cells is regulated by canonical Wnt-
Abbreviations: PSCs, porcine satellite cells; DEGs, differentially expressed genes; Poly(I:C), Polyinosinic:polycytidylic acid; dsRNA, Double-stranded Ribonucleic acid; FPKM, fragments per kilobase of transcript per million mapped reads; FC, fold change; cDNA, DNA complementary to RNA; qPCR, quantitative real-time PCR; ANOVA, analysis of variance; PBS, Phosphate buffered saline; KEGG, Kyoto Encyclopedia of Genes and Genomes; GO, Gene Ontology; BPs, biological processes; CCs, cellular components; MFs, molecular functions; DAPI, 4′,6-diamidino-2-phenylindole; MTT, 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide ⁎ Corresponding author at: Key Laboratory of Agricultural Animal Genetics, Breeding, and Reproduction, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan 430070, PR China. E-mail addresses: [email protected] (T.-N. Tran-Thi), [email protected] (S. Wang), [email protected] (A.A. Adetula), [email protected] (C. Zou), [email protected] (A.I. Omar), [email protected] (J.-L. Han), [email protected] (D.-X. Zhang), [email protected] (S.-H. Zhao). https://doi.org/10.1016/j.gene.2018.12.059 Received 7 July 2018; Received in revised form 21 December 2018; Accepted 30 December 2018 Available online 08 January 2019 0378-1119/ © 2019 Published by Elsevier B.V.
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paraformaldehyde for 15 min. Next, 0.3% Triton X-100 was added to PSCs for 10 min at room temperature. After that, blocking of unspecific binding of the antibody and permeabilization were performed using 3% bovine serum albumin, 0.3% Triton X-100 and 10% fetal bovine serum in PBS solution for 2 h at room temperature. PSCs were incubated with the primary anti-myosin (1:1000) at 4 °C overnight. The anti-mouse IgG was used as the secondary antibodies for 2 h at room temperature. Then, the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by the same Olympus inverted microscope system.
signalling pathway via the overexpression of Wnt1, Wnt3a and Wnt5a proteins (Otto et al., 2008). The regulation between self-renewal and differentiation of satellite cells is directed by Fas-associated death domain (Cheng et al., 2014). Polyinosinic:polycytidylic acid (poly(I:C)), a synthetic double stranded RNA (dsRNA), has been applied to mimic viral infection to induce the immune response models in experimental animals. Poly(I:C) induced an antiviral response in many cell types such as human corneal epithelial cells (Kumar et al., 2006) and lung endothelial cells (Huang et al., 2016), prompted the apoptosis of human CD34+ cells (Liu et al., 2012), but inhibited the differentiation of mouse preadipocytes (Yu et al., 2016). Therefore, in this study, we used poly(I:C) to induce the response of porcine satellite cells (PSCs) to a mimicking viral infection, for a depth understanding of the response mechanisms of PSCs. RNA-Seq is one of the next-generation sequencing technologies and is widely used for transcription profiling because of its high sensitivity, reproducibility, dynamic range of expression and base pair resolution. This technology was used to study the transcriptomic profiles of PSCs under proliferation and differentiation phases (Jensen et al., 2017). Considering the importance of satellite cells for the regeneration and development of animal skeletal muscle, we executed this study to examine the effect of poly(I:C) stimulation on PSCs during the proliferation and differentiation phases. This study has the potential to reveal novel genes and pathways responsible for immune barrier to self-protection of poly(I:C)-induced PSCs.
2.4. RNA quality control, sequencing and analysis A total of 21 RNA samples were isolated from PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation using the TRIzol Reagent according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNA purity was checked using the NanoPhotometer spectrophotometer (IMPLEN, CA, USA). RNA integrity was assessed using the RNA 6000 Nano assay of the Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., CA, USA). Twenty-one first strand cDNA libraries were synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNaseH-). The Solexa sequencing was implemented on the Illumina Hiseq 4000 Systems (NEB, USA). The raw sequence data files discussed in this experiment have been deposited in NCBI's Gene Expression Omnibus (GEO) (Series GSE112527) (https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE112527) (accession numbers included untreated PSCs at 0 h: GSM3072241, GSM3072242 and GSM3072243; untreated PSCs at 12 h: GSM3072244, GSM3072245 and GSM3072246; treated PSCs at 12 h: GSM3072247, GSM3072248 and GSM3072249; untreated PSCs at 24 h: GSM3072250, GSM3072251 and GSM3072252; treated PSCs at 24 h: GSM3072253, GSM3072254 and GSM3072255; untreated PSCs at 48 h: GSM3072256, GSM3072257 and GSM3072258; treated PSCs at 48 h: GSM3072259, GSM3072260 and GSM3072261). The quality of obtained sequencing data was controlled using the FastQC software (Babraham Bioinformatics, Cambridge, UK) and trimmed using the Trimmomatic tool (Bolger et al., 2014). The adapters were trimed, all reads were mapped to and sequences were identified based on the Ensembl Sus scrofa 11.1.90 set GTF annotation genome (available at ftp://ftp.ensembl.org/pub/release-90/gtf/sus_scrofa/) using the HISAT2 program (Kim et al., 2015). Afterwards, the Stringtie assembler was used to estimate gene expression and transcript levels via the FPKM (fragments per kilobase of transcript per million mapped reads) approach (Pertea et al., 2015). Thirteen comparison libraries were performed using the data obtained from the RNA-Seq, including untreated groups: 0 h vs 12 h untreated, 12 h vs 24 h untreated, 24 h vs 48 h untreated, 0 h vs 24 h untreated and 0 h vs 48 h untreated; and treated groups: 0 h vs 12 h treated, 12 h vs 24 h treated, 24 h vs 48 h treated, 0 h vs 24 h treated and 24 h vs 48 h treated; and between treated and untreated groups: 12 h untreated vs treated, 24 h untreated vs treated and 48 h untreated vs treated groups. Then, the differentially expressed genes (DEGs) among different comparison libraries were identified with q-value < 0.05 and absolute of log2 fold change (|log2FC|) ≥ 1.5 using the Ballgown package (Frazee et al., 2015).
2. Materials and methods 2.1. Porcine satellite cells and poly(I:C) stimulation All experimental procedures were strictly conducted following the guidelines established by the Standing Committee of Hubei People's Congress (No. 5) and approved by the Scientific Ethics Committee of Huazhong Agricultural University. PSCs were isolated from five-day -old piglet hind-limb muscles. The isolated PSCs were placed in 6-well plates and incubated in the growth media (Bovine serum, DMEM, Gentamycin and AA solution) at 37 °C and 5% CO2 until their confluence around 24 hours (h) of culture. Then, PSCs were treated with poly(I:C) at 10 μg/mL for 0, 12, 24, and 48 h while the untreated PSCs were included as negative controls. Next, PSCs were rinsed with phosphate-buffered saline (PBS) solution and induced to differentiate by the differentiation media containing 5% horse serum. The triplicate samples at 0, 12, 24 and 48 h of poly(I:C) stimulation were observed under an Olympus inverted microscope system (Olympus, Tokyo, Japan) (Fig. 2(A-H)) and then harvested using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) for further analyses. 2.2. Proliferation assay Cell viability was analyzed using 3-(4,5-dimethylthiazolyl-2)-2,5diphenyltetrazolium bromide (MTT) assay kit according to the manufacturer's instructions (Sigma Chemical Co.). Briefly, PSCs were cultured with 10 μg/mL poly(I:C) in a 96-well plate at a density of 2 × 104 cells per well. After 12 h of poly(I:C) stimulation, PSCs were incubated at 37 °C with 10 μL of MTT (5 mg/mL) solution for 4 h. Then, the medium was removed, and 100 μL of Formazan solvent was added into each well for 1 h at 37 °C. The absorbance at 570 nm was measured using an EnSpire Multimode Plate Reader (PerkinElmer). The percentage of the absorbance value was used to evaluate the cell viability.
2.5. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses The up- and down-regulated genes were separated to investigate the functions of genes and pathways, using the version of Sus scrofa annotation in Database for Annotation, Visualization, and Integrated Discovery platform (DAVID, https://david.ncifcrf.gov/). The GO terms (including biological processes (BPs), cellular components (CCs), and molecular functions (MFs)) were utilized to detect the functions of genes. The pathway detection was identified by the KEGG search pathway tools (Kanehisa and Goto, 2000).
2.3. Differentiation assay The differentiation of PSCs was observed by myosin immunofluorescence staining. Briefly, PSCs after proliferation were induced to differentiate in the differentiation media containing 5% horse serum. Then, PSCs were rinsed twice with PBS solution and fixed with 4% 114
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observations of PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation are illustrated in Fig. 2(A–H).
2.6. Quantitative RT-PCR (qPCR) and statistical analysis To investigate the levels of gene expressions after poly(I:C) stimulation, total RNA obtained from the samples was converted into cDNA using the PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) (TaKaRa Bio Inc., Otsu, Japan). The qPCR was performed using the SYBR® Green Real-time PCR Master Mix (Toyobo Co., Ltd., Osaka, Japan) on the CFX384 Touch™ Real-Time PCR Detection System following the manufacturer's instructions (Bio-Rad). The Oligo7 Primer Analysis software (Molecular Biology Insights, Inc., Cascade, CO, USA) was applied to design and evaluate the primers for gene validations. The specific primer sequences used for qPCR are shown in Supplementary file 1. The qPCR was performed as follows: 1 cycle at 95 °C for 2 min, 40 cycles at 95 °C for 30 s, 60 °C for 20 s and 72 °C for 15 s. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize the expression levels. SPSS 21.0 software was utilized to estimate qPCR datasets with one-way analysis of variance (ANOVA) test (Ganger et al., 2017). The significant differences were considered at p-value < 0.05.
3.2. RNA-Seq and differentially expressed genes (DEGs) A total of 13 comparison libraries of PSCs were analyzed from the data obtained by the RNA-Seq method, including untreated and treated PSCs at 0, 12, 24 and 48 h of poly(I:C) stimulation. The summary of the RNA-Seq data from PSCs are shown in Supplementary file 2. Interestingly, we found 406 DEGs from 4 out of the 13 comparison libraries (q-value < 0.05 and |log2fold-change| ≥ 1.5), including 88 DEGs in 0 h vs 12 h treated, 119 DEGs in 12 h vs 24 h treated, 104 DEGs in 0 h vs 24 h treated and 95 DEGs in 24 h vs 48 h untreated comparison libraries. The numbers and expression levels of DEGs identified in each pair of the comparison libraries (0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated) are shown in Fig. 3(A–C) and Supplementary file 3, among which 241 were upregulated but 165 were down-regulated. The highest level of an upregulated gene was 8.26-fold change (FC) in 12 h vs 24 h treated comparison, whereas the lowest level of a down-regulated gene was −6.48 FC in 0 h vs 12 h treated comparison (Fig. 3(C)). Venn diagrams were used to distinguish the overlapped and unique DEGs among upand down-regulated genes. Among up-regulated genes, 71 DEGs were unique in 0 h vs 12 h treated comparison libraries, whereas 15 DEGs were overlapped between 12 h vs 24 h treated and 0 h vs 24 h treated comparison libraries (Fig. 3(A)). Similarly, 20 down-regulated genes were overlapped in 12 h vs 24 h treated and in 0 h vs 24 h treated comparison libraries (Fig. 3(B) and Supplementary file 4). The hierarchically clustered heatmaps were constructed to classify the transcriptional profiles of the 406 DEGs among the comparison libraries of untreated and treated PSC groups (Fig. 4 and Fig. 5(A-D)). The overall heatmap for all samples illustrated the differences (qvalue < 0.05) between the groups and among poly(I:C) stimulations during the proliferation and differentiation phases (Fig. 4). During the proliferation phase, there was no significant difference (q-value ≥ 0.05) between the untreated groups at 0 h and 12 h of the stimulation, whereas there were significant differences (q-value < 0.05) between treated groups at 0 h and 12 h of the stimulation. Among the overall expression, four comparison libraries were identified with significant DEGs (Fig. 5(A-D)). The hierarchically clustered heatmaps showed
3. Results 3.1. Proliferation and differentiation phases of PSCs To study the gene expression levels at pre- and post-poly(I:C) stimulation during the proliferation and differentiation phases, we isolated and cultured PSCs from the piglet hind limb muscles. The cell visibility of PSCs during the proliferation phase was measured by MTT assay (Fig. 1(A)). The results showed that there was no significant difference in cell visibility between untreated (100%) and treated (94.43%) groups, suggesting that mimicking viral infection did not induce the death of PSCs during the proliferation phase. However, the cell fusion and myotube formation during the differentiation phase of PSCs were definitely distinguishable between untreated and treated groups when they were observed using the myosin immunofluorescence staining (Fig. 1(B)). During the differentiation stage, the cell fusion and myotubes are constructed by an arrangement of adjacent cells. The myosin immunofluorescence staining results indicated that the elongated shape of myotubes in the untreated groups was more visibly distinct than that in the treated groups (Fig. 1(B)). The microscope
Fig. 1. PSCs after poly(I:C) stimulation. (A) Cell viability after poly(I:C) stimulation during the proliferation phase. (B) Myosin immunofluorescence staining of PSCs during the differentiation phase. Nucleus was stained with DAPI (blue). Scale bars: 100 μm. Magnification: 100×. 115
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Fig. 2. Phase microscopy images of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation. Proliferation phase was performed within 12 h while differentiation phase was performed between 24 and 48 h of poly(I:C) stimulation. (A), (B), (C) and (D) are the untreated PSCs at 0, 12, 24 and 48 h of the stimulation, respectively. (E), (F), (G) and (H) are the treated PSCs at 0, 12, 24 and 48 h of the stimulation, respectively.
were associated with muscle contraction, muscle structure development, actin filament-based process and cytoskeleton regulation, while down-regulated genes were related to viral genome replication, immune response and response to external stimulus (Fig. 6(C-D)). In 0 h vs 24 h treated comparison libraries, up-regulated genes were enriched in BPs of the regulations of defense response, regulation of response to stress, regulations of cell communication and signal transduction (Fig. 6(E)); whereas cell cycle process, regulation cell cycle and mitotic cell cycle phase transition were represented among down-regulated genes (Fig. 6(F)). The DEGs involved in CCs and MFs were less than in BPs; in which, most up-regulated genes during the differentiation phase were engaged in the development of muscle, whereas down-regulated genes belonged to cellular processes (Supplementary file 5). The DEGs were mapped to canonical pathways based on KEGG database (Supplementary file 6 and Table 1). Interestingly, the significant pathways emerged in 0 h vs 12 h treated comparison libraries included Influenza A, proteasome, Hepatitis B and C, measle, and Herpes simplex infection pathways, RIG-I-like receptor, TLR and chemokine signaling pathways, and cytosolic DNA-sensing pathway (Table 1). The pathways related to the cell cycle and DNA replication in
significant differences (q-value < 0.05) in comparisons of 0 h vs 12 h treated libraries, 12 h vs 24 h treated libraries, 0 h vs 24 h treated libraries and 24 h vs 48 h untreated libraries (Fig. 5(A-D)). Therefore, these results distinguished the changes in gene expression levels of PSCs after poly(I:C) stimulation during the proliferation and differentiation phases. 3.3. GO and KEGG pathway analyses Functional classifications based on GO categories annotated the DEGs from comparison libraries. The GO term results are shown in Fig. 6(A-H) and Supplementary file 5. The representative collections of BPs were selected and depicted by both the level of enrichments and the number of genes. During the proliferation phase, up-regulated genes found in the BPs of 0 h vs 12 h treated comparison libraries were related to immune system such as regulation of innate immune response, immune effector processes and defense response to virus; whereas downregulated genes were associated with muscle organ development, muscle structure development and response to growth factor (Fig. 6(AB)). In 12 h vs 24 h treated comparison libraries, up-regulated genes
Fig. 3. The numbers and expression levels of DEGs among comparison libraries. Venn diagrams present the numbers overlapped of DEGs in comparison libraries (0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated groups). (A) and (B) Distribution of up- and down-regulated genes between the comparison libraries, respectively. (C) The average expression levels of DEGs between comparison libraries with Mean ± SEM. Error bars represent standard error (SEM) of the mean. 116
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Fig. 4. The overall heatmap of DEGs between the untreated and treated PSCs during the proliferation and differentiation phases. Each row represents a sample while each column represents a gene. The color key value shows the minimum to maximum (0 to 10) expression of genes by binary logarithms of (FPKM + 1).
(IFIH1), interferon-stimulated gene 15 (ISG15) and C-C motif chemokine ligand 5 (CCL5) related to immune response were highly expressed (p-value < 0.05) at 12 h of the stimulation, whereas their expression levels decreased between 24 and 48 h of the stimulation during the differentiation phase (p-value < 0.05). Besides, three genes including myosin heavy chain 4 (MYH4), leiomodin-3 (LMOD3) and tripartite motif containing 72 (TRIM72) related to muscle differentiation were also highly expressed (p-value < 0.05) during the differentiation phase, however, their expression levels were lower in treated than untreated groups. The validation results are shown in Fig. 7.
the comparison libraries of 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated groups are shown in Supplementary file 6. 3.4. Validation analysis We selected nine DEGs which were involved in signaling pathways and BPs in 0 h vs 12 h treated comparison libraries, to validate their expression levels uncovered from the RNA-Seq analysis. Six up-regulated genes including DExD/H-Box helicase 58 (DDX58), radical S adenosyl methionine domain containing 2 (RSAD2), 2′-5′-oligoadenylate synthetase 2 (OAS2), interferon induced with helicase C domain 1
Fig. 5. The DEGs of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation (A), (B), (C), (D) for the DEGs in 0 h vs 12 h treated, 12 h vs 24 h treated, 0 h vs 24 h treated and 24 h vs 48 h untreated comparison libraries, respectively. Each column represents a sample while each row represents a gene. The color key value shows the minimum to maximum (0 to 10) expression of genes by binary logarithms of (FPKM + 1). 117
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Fig. 6. The distinctive BPs of DEGs among comparison libraries. (A) and (B) for BPs of up- and down-regulations in 0 h vs 12 h treated comparison libraries, respectively; (C) and (D) for BPs of up- and down-regulations in 12 h vs 24 h treated comparison libraries, respectively; (E) and (F) for BPs of up- and downregulations in 0 h vs 24 h treated comparison libraries, respectively; (G) and (H) for BPs of up- and down-regulations in 24 h vs 48 h untreated comparison libraries, respectively. (UN: untreated; TR: treated; up: up-regulation; down: down-regulation).
4. Discussion
regulated genes were associated with protein impairment, growth and differentiation of PSCs (Jensen et al., 2012). However, the gene expression levels during viral infection of skeletal muscle satellite cells have not been unconcealed. In this study, we stimulated PSCs using poly(I:C) to induce immune responses and then performed the RNA-Seq analysis to determine DEGs and the signaling pathways of PSCs undergoing mimicking viral infection. Our results showed that after poly(I:C) stimulation, the clusters of up-regulated genes were associated with BPs related to defense and immune responses, including type I interferon (IFN-I) production (Supplementary file 5). Previous studies indicated that the mimicking
PSCs are not only known for their essential role in muscle formation and development, but also their ability of excellent self-renewal after muscle injury. The transcriptome profiling of skeletal muscle satellite cells during myotube formation revealed that the up-regulated genes were involved in the muscle contraction and muscle system process in several species such as pig, caprine and bovine (Tripathi et al., 2014; Tong et al., 2015; Jensen et al., 2017). In human, the skeletal muscle satellite cells were identified as the infectious target of Chikungunya virus (Ozden et al., 2007). In the acute physical stimulation, most up118
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Table 1 The signaling pathways identified in 0 h vs 12 h treated comparison libraries. Pathways
Terms
Gene count
p-value
Genes
Enrichment
ssc05164 ssc05168 ssc04622 ssc04623 ssc04620 ssc05160 ssc05162 ssc05161 ssc04062 ssc03050
Influenza A Herpes simplex infection RIG-I-like receptor signaling pathway Cytosolic DNA-sensing pathway Toll-like receptor signaling pathway Hepatitis C Measles Hepatitis B Chemokine signaling pathway Proteasome
9 8 6 5 5 5 5 4 4 3
1.78E−07 5.19E−06 6.04E−06 8.99E−05 6.30E−04 0.001486 0.001714 0.021484 0.030565 7.18E−04
DDX58, IFIH1, IRF7, RSAD2, OAS2, CCL5, STAT1, FURIN, CXCL10 DDX58, IFIH1, IFIT1, IRF7, TAP1, OAS2, CCL5, STAT1 DDX58, IFIH1, ISG15, IRF7, DHX58, CXCL10 DDX58, MB21D1, IRF7, CCL5, CXCL10 IRF7, CXCL9, CCL5, STAT1, CXCL10 DDX58, IFIT1, IRF7, OAS2, STAT1 DDX58, IFIH1, IRF7, OAS2, STAT1 DDX58, IFIH1, IRF7, STAT1 CXCL9, CCL5, STAT1, CXCL10 PSME1, PSMB8, PSMB9
13.06 10.66 21.39 19.87 12.00 9.54 9.18 6.42 5.60 21.55
trigged by mediated TLR3 signaling pathway (Ning et al., 2011). In this study, transcriptional factor of IRF7 gene participated in TLR signaling pathway, suggesting its significant role in dictating the outcome of innate immune responses. The expressed genes associated with TLR signaling pathway were also related to chemokine and transcription factor families (also see Medzhitov, 2001) (Table 1). Chemokines play an essential role in inflammatory responses, immune modulators and lymphopoiesis (Zlotnik and Yoshie, 2000). An early investigation demonstrated that dsRNA induced the secretion of proinflammatory chemokines such as CCL5 in the bronchial epithelial cells (Gern et al., 2003). In our results, several chemokine members such as CCL5, C-X-C motif chemokine ligand 9 (CXCL9) and C-X-C motif chemokine ligand 10 (CXCL10) were up-regulated during poly(I:C) stimulation and involved in chemokine signaling pathway. Thus, our finding proposed that mimicking viral poly(I:C) stimulation activated the expression of immune and inflammatory response genes through TLR and chemokine signaling pathways in PSCs. These pathways are representatives which were induced by poly(I:C) stimulation in many cell types (Gern et al., 2003; Yan et al., 2013; Yu et al., 2016). As mention above, the expressions of MYH4, LMOD3 and TRIM72 genes in treated group were lower than in the negative controls during the differentiation phase (24 to 48 h of the stimulation) (Fig. 7). The expression levels of marker genes such as LMOD3, myomesin 3 (MYOM3) and neurogenic locus notch homolog protein 3 (NOTCH3) associated with the muscle differentiation and self-renewal of muscle satellite cells were revealed in our study (Supplementary file 3), consistent with a recent study (Flamini et al., 2018). Previous studies demonstrated that MYH4 expression was high in fast skeletal muscle of pig (Brown et al., 2014). Besides, LMOD3 expression was induced in early differentiation period of skeletal muscle along with its strong actin filament (Yuen et al., 2014). In addition, the expression of TRIM72 gene also plays an important role in the differentiation period of skeletal muscle (Cai et al., 2009). All these lines of evidence indicated that the expression levels of MYH4, LMOD3 and TRIM72 genes in the myogenesis of skeletal muscle are crucial. Ultimately, we speculated that viral infection may affect the regulation of genes related to the regeneration and development of PSCs.
viral poly(I:C) stimulation induced innate immune response in adipocytes and suggested the indirect inhibition of preadipocyte differentiation through downstream signaling pathway (Yu et al., 2016). In our results, the expression levels of genes such as MYH4, LMOD3 and TRIM72 (Fig. 7) related to the differentiation of PSCs were reduced after poly(I:C) stimulation. Thus, our finding suggested that the mimicking virus may also inhibit the differentiation of PSCs, similar to the results of poly(I:C) treated adipocytes (Yu et al., 2016). After trauma injury, the expression of genes related to immune response, chemokine and inflammation were up-regulated in the early period, while the genes associated with proliferation and differentiation regulations were exhibited in the middle period (Aguilar et al., 2016). Similarly, we observed high expressions of DEGs related to immune response and viral processes during mimicking viral infection of PSCs in the early period. The changes in gene expression levels of PSCs after poly(I:C) stimulation were investigated via functional pathway analysis. We focused on the changes in 0 h vs 12 h treated comparison libraries because of remarkable signaling pathways related to immunity functions (Table 1 and Supplementary file 6). Poly(I:C) is recognized by the pattern recognition receptors of Toll-like receptor 3 (TLR3), retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5) (Yu and Levine, 2011). In the airway epithelial cells, poly (I:C) induced the up-regulation of IFN-I gene through stimulation of the RIG-I/MAVS pathway (Dauletbaev et al., 2015). Besides, poly(I:C) also stimulated the expression of antiviral effector gene 2′,5′-oligoadenylate synthetase (OAS) and IFN-I gene in human corneal epithelial cells (Kumar et al., 2006). In our results, the expressions of genes related to IFN-I, proinflammatory cytokines and chemokines such as DDX58, IFIH1 and CCL5 were increased (Table 1 and Fig. 7). Interestingly, these DEGs participated in influenza type A pathway, RIG-I-like receptor and TLR signaling pathways, and Hepatitis B and C pathways (Table 1). DDX58, also known as RIG-I, played a crucial role in the regulation of dsRNA signal induction and activated the expression of IFN-I gene (Yoneyama et al., 2004). Also, RIG-I expression was induced by a variety of viruses such as porcine reproductive and respiratory syndrome (Zhang et al., 2000), influenza, measles, Ebola, vesicular stomatitis and hepatitis C (Loo and Gale Jr., 2011). The activation of RIG-I and IFN-I genes were stimulated via a nucleotide component in panhandle promoter region of influenza A virus (Liu et al., 2015). In the present study, mimicking viral poly(I:C) stimulation induced RIG-I expression in most signaling pathways such as influenza A, herpes simplex infection, RIG-I-like receptor, cytosolic DNA-sensing, measles, hepatitis B and C, reflecting a significant function of RIG-I in immune regulation. Additionally, TLR and chemokine signaling pathways were activated by poly(I:C) stimulation in our study. TLRs play important roles in the recognition of pathogen-associated molecular patterns in innate immune system (Kawasaki and Kawai, 2014). Of which, TLR3 recognizes dsRNA and its analog (e.g. poly(I:C)) (Lester and Li, 2014). The activities of several interferon regulatory factors (IRFs) were regulated by TLR signaling pathways. Specifically the activation of IRF7 gene was
5. Conclusions In summary, we performed an RNA-Seq analysis to investigate the DEGs of PSCs during the proliferation and differentiation phases after poly(I:C) stimulation. We identified a number of DEGs related to immune system, regeneration and development of muscle. Influenza A, TLR and chemokine signaling pathways were identified in the poly(I:C) stimulation groups when compared to the untreated groups. Consequently, it is suggested that genes detected in this study are candidates for further analysis of regulatory mechanism of mimicking viral induced PSCs. Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2018.12.059. 119
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Fig. 7. Validations of DEGs by qPCR analysis between untreated and treated PSCs. Blue bars represent the untreated PSCs while red bars represent the treated PSCs at 0, 12, 24 and 48 h of the stimulation. GAPDH is used as a housekeeping gene for normalizing their expression levels. Six genes (DDX58, RSAD2, OAS2, IFIH1, ISG15 and CCL5) were highly expressed in the treated PSCs at 12 h of the stimulation while three genes (MYH4, LMOD3 and TRIM72) were highly expressed in the untreated PSCs when compared to the treated PSCs during the differentiation phase (between 24 and 48 h). * Significant level at p-value < 0.05 is based on Fishers least significant difference post hoc test of one-way ANOVA analysis. Error bars represent standard error (SEM) of the mean.
Author contributions
China (CGIAR 31361140365 and 31372302), the National High Technology Plan of China (2013AA102502) and the Fund of Modern Industrial Technology System of Pig (CARS-35).
SHZ, JLH, SW and TNTT designed the experiments; TNTT and SW cultured and stimulated PSCs by poly(I:C); AIO and TNTT implemented qPCR experiments; DXZ, AAA and CZ analyzed data; TNTT wrote the manuscript; SHZ, JLH, AAA and TNTT revised manuscript. All authors read and approved the final manuscript.
Acknowledgements We would like to acknowledge Professor Xinyun Li for his precious advice and encouragement during the implementation of the experiment.
Funding This work was supported by National Natural Science Foundation of 120
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Conflicts of interest
synthetic double-stranded RNA, poly I:C, induces a rapid apoptosis of human CD34(+) cells. Exp. Hematol. 40, 330–341. Liu, G., Park, H.-S., Pyo, H.-M., Liu, Q., Zhou, Y., 2015. Influenza A virus panhandle structure is directly involved in RIG-I activation and interferon induction. J. Virol. 89, 6067–6079. Loo, Y.M., Gale Jr., M., 2011. Immune signaling by RIG-I-like receptors. Immunity 34, 680–692. Mauro, A., 1961. Satellite cell of skeletal muscle fibers. J. Biophys. Biochem. Cytol. 9, 493–495. Medzhitov, R., 2001. Toll-like receptors and innate immunity. Nat. Rev. Immunol. 1, 135–145. Nierobisz, L.S., Mozdziak, P.E., 2008. Factors influencing satellite cell activity during skeletal muscle development in avian and mammalian species. Asian Australas. J. Anim. Sci. 21, 456–464. Ning, S., Pagano, J.S., Barber, G.N., 2011. IRF7: Activation, regulation, modification and function. Genes Immun. 12, 399–414. Otto, A., Schmidt, C., Luke, G., Allen, S., Valasek, P., Muntoni, F., Lawrence-Watt, D., Patel, K., 2008. Canonical Wnt signalling induces satellite-cell proliferation during adult skeletal muscle regeneration. J. Cell Sci. 121, 2939–2950. Ozden, S., Huerre, M., Riviere, J.P., Coffey, L.L., Afonso, P.V., Mouly, V., de Monredon, J., Roger, J.C., El Amrani, M., Yvin, J.L., Jaffar, M.C., Frenkiel, M.P., Sourisseau, M., Schwartz, O., Butler-Browne, G., Despres, P., Gessain, A., Ceccaldi, P.E., 2007. Human muscle satellite cells as targets of Chikungunya virus infection. PLoS One 2, e527. Pertea, M., Pertea, G.M., Antonescu, C.M., Chang, T.-C., Mendell, J.T., Salzberg, S.L., 2015. Stringtie enables improved reconstruction of a transcriptome from RNA-seq reads. Nat. Biotechnol. 33, 290–295. Relaix, F., Zammit, P.S., 2012. Satellite cells are essential for skeletal muscle regeneration: The cell on the edge returns centre stage. Development 139, 2845–2856. Shi, X., Garry, D.J., 2006. Muscle stem cells in development, regeneration, and disease. Genes Dev. 20, 1692–1708. Tong, H.L., Yin, H.Y., Zhang, W.W., Hu, Q., Li, S.F., Yan, Y.Q., Li, G.P., 2015. Transcriptional profiling of bovine muscle-derived satellite cells during differentiation in vitro by high throughput RNA sequencing. Cell. Mol. Biol. Lett. 20, 351–373. Tripathi, A.K., Patel, A.K., Shah, R.K., Patel, A.B., Shah, T.M., Bhatt, V.D., Joshi, C.G., 2014. Transcriptomic dissection of myogenic differentiation signature in caprine by RNA-Seq. Mech. Dev. 132, 79–92. Yan, K., Zhu, W., Yu, L., Li, N., Zhang, X., Liu, P., Chen, Q., Chen, Y., Han, D., 2013. Tolllike receptor 3 and RIG-I-like receptor activation induces innate antiviral responses in mouse ovarian granulosa cells. Mol. Cell. Endocrinol. 372, 73–85. Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira, K., Akira, S., Fujita, T., 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5, 730–737. Yu, M., Levine, S.J., 2011. Toll-like receptor, RIG-I-like receptors and the NLRP3 inflammasome: Key modulators of innate immune responses to double-stranded RNA viruses. Cytokine Growth Factor Rev. 22, 63–72. Yu, L., Liu, G., Yang, C., Song, X., Wang, H., 2016. Polyinosinic-polycytidylic acid inhibits the differentiation of mouse preadipocytes through pattern recognition receptormediated secretion of tumor necrosis factor-alpha. Immunol. Cell Biol. 94, 875–885. Yuen, M., Sandaradura, S.A., Dowling, J.J., Kostyukova, A.S., Moroz, N., Quinlan, K.G., Lehtokari, V.L., Ravenscroft, G., Todd, E.J., Ceyhan-Birsoy, O., Gokhin, D.S., Maluenda, J., Lek, M., Nolent, F., Pappas, C.T., Novak, S.M., D'Amico, A., Malfatti, E., Thomas, B.P., Gabriel, S.B., Gupta, N., Daly, M.J., Ilkovski, B., Houweling, P.J., Davidson, A.E., Swanson, L.C., Brownstein, C.A., Gupta, V.A., Medne, L., Shannon, P., Martin, N., Bick, D.P., Flisberg, A., Holmberg, E., Van den Bergh, P., Lapunzina, P., Waddell, L.B., Sloboda, D.D., Bertini, E., Chitayat, D., Telfer, W.R., Laquerriere, A., Gregorio, C.C., Ottenheijm, C.A., Bonnemann, C.G., Pelin, K., Beggs, A.H., Hayashi, Y.K., Romero, N.B., Laing, N.G., Nishino, I., Wallgren-Pettersson, C., Melki, J., Fowler, V.M., MacArthur, D.G., North, K.N., Clarke, N.F., 2014. Leiomodin-3 dysfunction results in thin filament disorganization and nemaline myopathy. J. Clin. Invest. 124, 4693–4708. Zhang, X., Wang, C., Schook, L.B., Hawken, R.J., Rutherford, M.S., 2000. An RNA helicase, RHIV-1, induced by porcine reproductive and respiratory syndrome virus (PRRSV) is mapped on porcine chromosome 10q13. Microb. Pathog. 28, 267–278. Zlotnik, A., Yoshie, O., 2000. Chemokines: A new classification system and their role in immunity. Immunity 12, 121–127.
The authors declare that they have no conflict of interest. References Aguilar, Carlos A., Pop, R., Shcherbina, A., Watts, A., Matheny Jr., Ronald W., Cacchiarelli, D., Han, Woojin M., Shin, E., Nakhai, Shadi A., Jang, Young C., Carrigan, Christopher T., Gifford, Casey A., Kottke, Melissa A., Cesana, M., Lee, J., Urso, Maria L., Meissner, A., 2016. Transcriptional and chromatin dynamics of muscle regeneration after severe trauma. Stem Cell Rep. 7, 983–997. Boldrin, L., Muntoni, F., Morgan, J.E., 2010. Are human and mouse satellite cells really the same? J. Histochem. Cytochem. 58, 941–955. Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. Briata, P., Lin, W.J., Giovarelli, M., Pasero, M., Chou, C.F., Trabucchi, M., Rosenfeld, M.G., Chen, C.Y., Gherzi, R., 2012. PI3K/AKT signaling determines a dynamic switch between distinct KSRP functions favoring skeletal myogenesis. Cell Death Differ. 19, 478–487. Brown, D.M., Brameld, J.M., Parr, T., 2014. Expression of the myosin heavy chain IIB gene in porcine skeletal muscle: The role of the CArG-Box promoter response element. PLoS One 9, e114365. Cai, C., Masumiya, H., Weisleder, N., Pan, Z., Nishi, M., Komazaki, S., Takeshima, H., Ma, J., 2009. MG53 regulates membrane budding and exocytosis in muscle cells. J. Biol. Chem. 284, 3314–3322. Cheng, W., Wang, L., Yang, B., Zhang, R., Yao, C., He, L., Liu, Z., Du, P., Hammache, K., Wen, J., Li, H., Xu, Q., Hua, Z., 2014. Self-renewal and differentiation of muscle satellite cells are regulated by the Fas-associated death domain. J. Biol. Chem. 289, 5040–5050. Dauletbaev, N., Cammisano, M., Herscovitch, K., Lands, L.C., 2015. Stimulation of the RIG-I/MAVS pathway by Polyinosinic:Polycytidylic acid upregulates IFN-β in airway epithelial cells with minimal costimulation of IL-8. J. Immunol. 195, 2829–2841. Flamini, V., Ghadiali, R.S., Antczak, P., Rothwell, A., Turnbull, J.E., Pisconti, A., 2018. The satellite cell niche regulates the balance between myoblast differentiation and self-renewal via p53. Stem Cell Rep. 10, 970–983. Frazee, A.C., Pertea, G., Jaffe, A.E., Langmead, B., Salzberg, S.L., Leek, J.T., 2015. Ballgown bridges the gap between transcriptome assembly and expression analysis. Nat. Biotechnol. 13, 243–246. Ganger, M.T., Dietz, G.D., Ewing, S.J., 2017. A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments. BMC Bioinf. 18, 534. Gern, J.E., French, D.A., Grindle, K.A., Brockman-Schneider, R.A., Konno, S., Busse, W.W., 2003. Double-stranded RNA induces the synthesis of specific chemokines by bronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 28, 731–737. Huang, L.Y., Stuart, C., Takeda, K., D'Agnillo, F., Golding, B., 2016. Poly(I:C) induces human lung endothelial barrier dysfunction by disrupting tight junction expression of claudin-5. PLoS One 11, e0160875. Jensen, J.H., Conley, L.N., Hedegaard, J., Nielsen, M., Young, J.F., Oksbjerg, N., Hornshoj, H., Bendixen, C., Thomsen, B., 2012. Gene expression profiling of porcine skeletal muscle in the early recovery phase following acute physical activity. Exp. Physiol. 97, 833–848. Jensen, J.H., Madsen, L.B., Panitz, F., Hornshøj, H., Nielsen, R.O., Bendixen, C., Oksbjerg, N., Thomsen, B., 2017. Transcriptome dynamics during proliferation and differentiation of porcine primary satellite cells. Gene Rep. 7, 169–175. Kanehisa, M., Goto, S., 2000. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30. Kawasaki, T., Kawai, T., 2014. Toll-like receptor signaling pathways. Front. Immunol. 5, 461. Kim, D., Langmead, B., Salzberg, S.L., 2015. HISAT: A fast spliced aligner with low memory requirements. Nat. Methods 12, 357–360. Kumar, A., Zhang, J., Yu, F.S., 2006. Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells. Immunology 117, 11–21. Lester, S.N., Li, K., 2014. Toll-like receptors in antiviral innate immunity. J. Mol. Biol. 426, 1246–1264. Liu, J., Guo, Y.M., Hirokawa, M., Iwamoto, K., Ubukawa, K., Michishita, Y., Fujishima, N., Tagawa, H., Takahashi, N., Xiao, W., Yamashita, J., Ohteki, T., Sawada, K., 2012. A
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