Characterization of Critical Functions of Long Non-Coding RNAs and mRNAs in Rhabdomyosarcoma Cells and Mouse Skeletal Muscle Infected by Enterovirus 71 Using RNA-Seq

Figures

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  Figure 1. Flow chart for novel lncRNA identification and classification. RD = rhabdomyosarcoma. EV71 = Enterovirus 71. hpi = hours post-infection. dpi = days post-infection. lncRNAs = long non-coding RNAs. The CPC, CPAT, CNCI and HMMer+ Pfam are software for predicting the coding ability of transcripts.

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  Figure 2. In vitro and in vivo models of EV71 infection. (A) Cytopathic effects in RD cells were captured via light microscopy (amplification: 100×) at 24 h post infection. (B) EV71 VP1 distribution in RD cells was imaged via confocal microscopy (amplification: 400×) at 24 hpi. (C) Clinical presentation in neonatal mice following EV71 infection. (D) Histopathological examination and EV71 VP1 distribution (brown) of skeletal muscle in mice at 7 days post infection by haematoxylin and eosin (H&E) staining and immunohistochemistry (IHC) staining, respectively. Red arrows represent inflammatory cell infiltration and necrotizing myositis with rupture of muscle fibers, and blue arrows represent VP1 positive staining area in skeletal muscle of mice. Bar=50 μm. DAPI = 4′, 6-diamidino-2-phenylindole.

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  Figure 3. Identification and classification of newly predicted lncRNAs. Venn diagram of the protein-coding ability prediction results in RD cells (A) and mice skeletal muscle (B). lncRNA classification annotation statistical graph for RD cells (C) and skeletal muscle of mice (D).

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  Figure 4. Heat maps of differentially-expressed gene expression ratios (log2FC) in EV71-infected human RD cells and skeletal muscle of neonatal mice. Differentially-expressed lncRNAs in RD cells (A) and skeletal muscle of mice (B) after EV71 infection. Differential expression profiles of mRNAs in RD cells (C) and skeletal muscle of mice (D). Red and green color denotes high relative expression and low relative expression, respectively.

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  Figure 5. Co-expression network of differentially-expressed lncRNA-mRNA based on Pearson’s correlation coefficient. (A) Significant differentially-expressed lncRNAs (23) and the top 50 correlated mRNAs with 685 connection edges in EV71-infected RD cells. (B) The top 50 differentially-expressed lncRNAs and their 50 most frequently altered relative mRNAs with 2490 connection edges in EV71-infected skeletal muscle of mice. The red node denotes lncRNA and the surrounding blue node represents mRNA.

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  Figure 6. Gene ontology (GO) analysis for differentially-expressed genes upon EV71 infection. (A–C) The top 10 significantly enriched GO terms in differential expression of mRNA, cis- and trans-acting target mRNAs in EV71-infected RD cells, respectively. (D–F) The top 10 significant GO terms in differentially-expressed mRNA, cis- and trans-regulated genes in EV71-infected skeletal muscle of mice, respectively.

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  Figure 7. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially-expressed genes by EV71 infection. (A–C) The top 20 enriched pathways associated with differentially-expressed mRNAs, cis- and trans-acting target genes in EV71-infected RD cells, respectively. (D–F) The top 20 associated pathways in differentially-expressed mRNAs, cis- and trans- regulated mRNAs in EV71-infected skeletal muscle of mice, respectively.

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  Figure 8. Comparisons of qPCR with RNA-seq results. (A) The validation results of qPCR. (B) The primary results of RNA-seq.