Transcriptional Analysis of lncRNA and Target Genes Induced by Influenza A Virus Infection in MDCK Cells

Abstract

Background: The MDCK cell line is the primary cell line used for influenza vaccine production. Using genetic engineering technology to change the expression and activity of genes that regulate virus proliferation to obtain high-yield vaccine cell lines has attracted increasing attention. A comprehensive understanding of the key genes, targets, and molecular mechanisms of viral regulation in cells is critical to achieving this goal, yet the post-transcriptional regulation mechanism involved in virus proliferation-particularly the effect of lncRNA on influenza virus proliferation-is still poorly understood. Therefore, this study used high-throughput RNA-seq technology to identify H1N1 infection-induced lncRNA and mRNA expression changes in MDCK cells and explore the regulatory relationship between these crucial lncRNAs and their target genes.

Results: In response to H1N1 infection in MDCK cells 16 h post-infection (hpi) relative to uninfected controls, we used multiple gene function annotation databases and initially identified 31,501 significantly differentially expressed (DE) genes and 39,920 DE lncRNAs (|log2FC| > 1, p < 0.05). Among these, 102 lncRNAs and 577 mRNAs exhibited predicted correlations with viral response mechanisms. Based on the magnitude of significant expression differences, related research, and RT-qPCR expression validation at the transcriptional level, we further focused on 18 DE mRNAs and 32 DE lncRNAs. Among these, the differential expression of the genes RSAD2, CLDN1, HCLS1, and IFIT5 in response to influenza virus infection was further verified at the protein level using Western blot technology, which showed results consistent with the RNA-seq and RT-qPCR findings. We then developed a potential molecular regulatory network between these four genes and their six predicted lncRNAs.

Conclusions: The results of this study will contribute to a more comprehensive understanding of the molecular mechanism of host cell non-coding RNA-mediated regulation of influenza virus replication. These results may also identify methods for screening target genes in the development of genetically engineered cell lines capable of high-yield artificial vaccine production.

Keywords: IAV; MDCK; influenza vaccine; lncRNAs; transcriptomics.

Figure 1

RT-qPCR was used to detect the expression changes of NP and NS1 genes of the A (H1N1) virus at different infection time points (12 hpi, 16 hpi, 24 hpi, 36 hpi) on MDCK + H1N1 cells. The data are expressed as x ± s (n = 3). ***: p < 0.001.

Figure 2

DE mRNAs and lncRNAs in MDCK cells infected with A(H1N1). MDCK cells were infected with A(H1N1) (X-275 strain; MOI = 0.01) or sham-infected with DMEM and harvested 16 hpi for analysis. Volcano plots of DE (A) mRNAs and (B) lncRNAs in MDCK + H1N1 cells relative to uninfected MDCK cells. (C) Heat map of DE mRNAs in MDCK + H1N1 cells relative to uninfected MDCK cells; each column represents a different sample, and each row represents a different mRNA. (D) Heat map of DE lncRNAs in MDCK + H1N1 cells relative to uninfected MDCK cells; each column represents a different sample, and each row represents a different lncRNA. Transcripts were considered significantly different using the following criteria: |log2FC| > 1 and p < 0.05. Abbreviations: DE, differentially expressed; IAV, influenza A virus; hpi, hours post-infection; MDCK + H1N1, infected MDCK cells; FC, fold change.

Figure 3

Screening of DE mRNAs related to virus response based on functional enrichment analysis. (A) Venn diagram of intersection analysis between virus-response related DE mRNAs in MDCK cells and predicted matched lncRNAs. Blue represents the 577 preliminarily screened virus response-related DE mRNAs; pink represents the predicted target gene (mRNA) of the total DE lncRNAs, and dark red (middle) represents DE mRNAs related to virus response and matching predicted DE lncRNAs. (B) GO function enrichment analysis of the 577 selected DE mRNAs. (C) Bubble diagram of the KEGG pathway enrichment analysis of the 577 preliminarily screened DE mRNAs, showing the top 20 KEGG pathways with the most significant enrichment. Abbreviations: DE, differentially expressed; GO, Gene Ontology.

Figure 4

Verification of DE mRNA expression using RT-qPCR in A(H1N1)-infected MDCK cells. RT-qPCR was performed on 18 screened DEGs identified from RNA-seq data in MDCK cells infected with A(H1N1) 16 hpi relative to uninfected MDCK cells. Blue represents MDCK cells, and white represents MDCK + H1N1 cells. Relative expression was determined using the 2^−ΔΔCt method with GAPDH as the internal reference gene. The data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using GraphPad Prism 8.0. *** p < 0.001. Abbreviations: DE, differentially expressed; IAV, influenza A virus; DEGs, differentially expressed genes; hpi, hours post-infection; MDCK + H1N1, infected MDCK cells.

Figure 5

Verification of DE lncRNA expression using RT-qPCR in A(H1N1)-infected MDCK cells. RT-qPCR was performed on 37 screened DE lncRNAs identified from RNA-seq data in MDCK cells infected with A(H1N1) 16 hpi relative to uninfected MDCK cells. Blue represents MDCK cells, and white represents MDCK + H1N1 cells. Relative expression was determined using the 2^−ΔΔCt method with GAPDH as the internal reference gene. The data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using GraphPad Prism 8.0. ** p < 0.01; *** p < 0.001. Abbreviations: DE, differentially expressed; IAV, influenza A virus; hpi, hours post-infection; MDCK + H1N1, infected MDCK cells.

Figure 6

Verification of CLDN1, HCLS1, IFIT5, and RSAD2 differential protein expression in A(H1N1)-infected MDCK cells. Western blot was performed on four screened DEGs in MDCK cells infected with A(H1N1) 16 hpi relative to uninfected MDCK cells. (A) SDS-PAGE gel and (B) quantitative protein expression levels are shown. GAPDH was used as the reference protein. Blue represents MDCK cells, and white represents MDCK + H1N1 cells. The data are expressed as mean ± standard deviation (n = 3). Statistical significance was determined using GraphPad Prism 8.0. ** p < 0.01; *** p < 0.001. Abbreviations: IAV, influenza A virus; DEGs, differentially expressed genes; hpi, hours post-infection; MDCK + H1N1, infected MDCK cells.

Figure 7

Interaction network of genes and co-expressed lncRNAs involved in IAV replication regulation in MDCK cells. Pink represents genes, and green represents lncRNA. RSAD2, CLDN1, IFIT5, and HCLS1 exhibit consistent expression patterns with their predicted matching lncRNAs, indicating that they may have a positive regulatory target gene relationship with their matching lncRNAs. RSAD2 and IFIT5 may inhibit IAV replication in MDCK cells through the type I/II interferon signaling pathway, in which RSAD2 inhibits the release of IAV from the plasma membrane of host cells. CLDN1, a key factor involved in HCV entry, may also assist in IAV entry into MDCK cells. HAX-1 interacts with the PA subunit of IAV, which may promote the replication of IAV in MDCK cells. Abbreviation: IAV, influenza A virus; HCV, hepatitis C virus; HAX-1, HCLS1 interacting protein X-1.

Figure 8

The expression levels of four mRNA (CLDN1, HCLS1, IFIT5, RSAD2) and their matching six lncRNAs (LTCONS-00127643, LTCONS-00140527, LTCONS-00139462, LTCONS-00139463, LTCONS-00085754, LTCONS-00085755) were detected using RT qPCR at different infection times (12 hpi, 16 hpi, 24 hpi) in MDCK + H1N1 cells. The data are expressed as x ± s (n = 3). *: p < 0.05; **: p < 0.01; ***: p < 0.001 (b).

Figure 9

The expression levels of four mRNA (CLDN1, HCLS1, IFIT5, RSAD2) and their matching six lncRNAs (LTCONS-00127643, LTCONS-00140527, LTCONS-00139462, LTCONS-00139463, LTCONS-00085754, LTCONS-00085755) were detected using RT qPCR when infected with different virus strains (A (H1N1), BY, VSV) on MDCK cells at 16 hpi. * p < 0.05; ** p < 0.01; *** p < 0.001; ns (no significance).BY: Influenza B/Phuket/3073/2013-like virus B (Yamagata/16/88 lineage); VSV: Vesicular stomatitis virus.