Deep Insight Into Long Non-coding RNA and mRNA Transcriptome Profiling in HepG2 Cells Expressing Genotype IV Swine Hepatitis E Virus ORF3

Abstract

Swine hepatitis E (swine HE) is a new type of zoonotic infectious disease caused by the swine hepatitis E virus (swine HEV). Open reading frame 3 (ORF3) is an important virulent protein of swine HEV, but its function still is mainly unclear. In this study, we generated adenoviruses ADV4-ORF3 and ADV4 negative control (ADV4-NC), which successfully mediated overexpression of enhanced green fluorescent protein (EGFP)-ORF3 and EGFP, respectively, in HepG2 cells. High-throughput sequencing was used to screen for differentially expressed long non-coding RNAs (lncRNAs) and messenger RNAs (mRNAs). The cis-target genes of lncRNAs were predicted, functional enrichment (Gene Ontology [GO] and Kyoto Encyclopedia of Genes and Genomes [KEGG]) was performed, and 12 lncRNAs with statistically significant different expressions (p ≤ 0.05 and q ≤ 1) were selected for further quantitative real-time reverse transcription (qRT-PCR) validation. In HepG2 cells, we identified 62 significantly differentially expressed genes (DEGs) (6,564 transcripts) and 319 lncRNAs (124 known lncRNAs and 195 novel lncRNAs) that were affected by ORF3, which were involved in systemic lupus erythematosus, Staphylococcus aureus infection, signaling pathways pluripotency regulation of stem cells, the peroxisome proliferator-activated receptor (PPAR) signaling pathway, and platinum drug resistance pathways. Cis-target gene prediction identified 45 lncRNAs corresponding to candidate mRNAs, among which eight were validated by qRT-PCR: LINC02476 (two transcripts), RAP2C-AS1, AC016526, AL139099, and ZNF337-AS1 (3 transcripts). Our results revealed that the lncRNA profile in host cells affected by ORF3, swine HEV ORF3, might affect the pentose and glucuronate interconversions and mediate the formation of obstructive jaundice by influencing bile secretion, which will help to determine the function of ORF3 and the infection mechanism and treatment of swine HE.

Keywords: HepG2; ORF3; lncRNA; swine HE; swine HEV.

Figure 1

Adenovirus of ADV4-ORF3 and ADV4-NC mediated overexpression of ORF3 in HepG2 cells. (A) Fluorescence microscope observation of HepG2 cells infected by adenovirus of ADV4-ORF3 and ADV4-NC for 24 h. (B) qRT-PCR validation for the relative expression level of ORF3. (C) Western blot analysis of HepG2 cells infected by adenovirus of ADV4-ORF3 and ADV4-NC for 24 h with rabbit polyclonal antibody against ORF3. β-Actin was used as an internal control. *p < 0.05, **p < 0.01.

Figure 2

Analysis of DEGs and transcripts. (A) Distribution of gene expression values for each sample. The x-axis is the sample name, and the y-axis is log10 (FPKM). The box graph of each region corresponds to five statistics (the maximum, the upper quartile, the median, the lower quartile, and the minimum, respectively, from the top to the bottom). (B) Gene expression density distribution. The x-axis is the log10 (FPKM), and the y-axis shows the gene expression density. (C) Volcano analysis of differential expression levels of genes; the x-axis is log2 (fold change), which represents the variation of the differential expression of multiple genes in different samples, and the y-axis shows the –log10 (p-value), which represents the statistical significance of the change in the gene expression levels; red indicates upregulated DEGs, and dark blue indicates downregulated DEGs. (D) The statistics of the frequency of upregulation and downregulation of genes with significantly differential expressions. Red represents upregulated genes, and dark blue represents downregulated genes. (E) The statistics of the frequency of upregulated and downregulated transcripts with significant differential expression. Red represents upregulated transcripts, and dark blue represents downregulated transcripts. (F) Heatmap of partial DEGs; red indicates upregulated DEGs, and dark blue indicates downregulated DEGs.

Figure 3

Analysis of differentially expressed lncRNAs. (A) Pie chart of different class code proportions of the lncRNAs in each sample. There are five class codes of lncRNA: j indicates a potentially novel isoform (fragment); i indicates a transfragment falling entirely within a reference intron; o indicates a generic exonic overlap with a reference transcript; u indicates an unknown, intergenic transcript; and x indicates an exonic overlap with the reference on the opposite strand. (B) Visualization results of lncRNAs from different samples, showing the distribution of lncRNA candidates in the chromosome more intuitively. (C) Volcano analysis of differentially expressed lncRNA levels; the x-axis is log2 (fold change), and the y-axis is –log10 (p-value). Red represents upregulated lncRNAs, and dark blue represents downregulated lncRNAs. (D) Statistics of the frequency of upregulated and downregulated lncRNAs with significant differential expression. Red represents upregulated lncRNAs, and dark blue represents downregulated lncRNAs. (E) Heatmap of differentially expressed lncRNAs; red represents upregulated lncRNAs, and dark blue represents downregulated lncRNAs.

Figure 4

Analysis of the structural characteristics of differentially expressed lncRNAs and mRNAs. (A) lncRNA and mRNA length statistics and comparisons; the x-axis represents the transcript length, and the y-axis represents the proportion. (B) Distribution of the ORF lengths of lncRNAs. (C) Distribution of the ORF lengths of mRNAs. (D) Statistics of lncRNA and mRNA exons. The x-axis represents the exon number of the lncRNA and mRNA, and the y-axis represents the proportion. (E) lncRNA and mRNA expression levels were statistically analyzed from two levels of log10 (FPKM) and number.

Figure 5

Analysis of the functional enrichment of target genes of the significantly differentially expressed lncRNAs. (A) GO functional enrichment analysis of the predicted target genes of the differentially expressed lncRNAs. y-axis: GO terms; x-axis: rich factor. The color of each bubble represents the p-value, and the bubble size represents the gene number. (B) KEGG pathway enrichment analysis of the predicted target genes of the differentially expressed lncRNAs. y-axis: pathway name; x-axis: rich factor. The color of each bubble represents the p-value, and the bubble size represents the gene number.

Figure 6

qRT-PCR validation for the eight selected significantly differentially expressed lncRNAs. *p < 0.05, **p < 0.01.