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To determine the propagation kinetics of H3N2 in A549 cells, we measured the viral titers at various time points after infection. Cells were infected with H3N2 at an MOI of 10 and then monitored by IFA at 12, 18, 24, and 36 hpi. The viral titer gradually increased at 12, 18, and 24 hpi and reached a maximum (105.7 TCID50/mL) at 24 hpi (Supplementary Fig. S1A and S1B). Based on these results, we selected cells infected with an MOI of 10 after 24 hpi for transcriptome analysis.
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High-throughput RNA-seq was performed to determine the expression levels of lncRNAs in A549 cells infected with H3N2 or uninfected (mock). More than 258 billion raw base reads were generated for each sample using an Illumina Hiseq platform. After removing adaptor and lowquality sequences, average size of each clean read was 135 nt, and the clean data Q30 was above 93.45% (Supplementary Table S2).
Based on various criteria, including the specific location in genome, multiple exons, length greater than 200 nt, and noncoding characteristics, transcripts were filtered by three steps to identify the annotated and novel lncRNAs. In total, 3031transcriptswere identifiedasnovel lncRNAs(Fig. 1A).
Figure 1. Identification of novel lncRNAs in H3N2-infected or non-infected cells. A LncRNA screening statistics in H3N2-infected or non-infected groups. B Evaluating the coding potential using four tools. C Classification of lncRNAs based on genomic location.
Next, protein-coding or noncoding transcripts were classified using four tools, i.e., CPAT, PLEK, CNCI, and CPC (Fig. 1B). Additionally, according to the corresponding genomic locations of transcripts of known protein-coding genes, newly identified lncRNAs were categorized into four groups, i.e., intronic lncRNAs (82%), intergenic lncRNAs (12%), antisense lncRNAs (3%), and bidirectional lncRNAs (3%; Fig. 1C).
Hierarchical clustering was performed to analyze the lncRNA expression profiles in H3N2-infected or noninfected cells. Obviously, expression levels of lncRNAs were significantly altered after H3N2 infection (Fig. 2A). In total, 6129 lncRNAs were differentially expressed, including 4963 upregulated lncRNAs and 1166 downregulated lncRNAs (fold change [FC] ≥ 2, P ≤ 0.05; Fig. 2B) (Supplementary Table S3). Of the differentially regulated lncRNAs, 22 newly identified lncRNAs were altered, with log2(FC) values of more than 12, compared with the noninfected group. The lncRNA showing the greatest upregulation was MSTRG.18254.3, with a log2(FC) of more than 16; in contrast, the lncRNA showing the greatest downregulation was MSTRG.11930.8, with a log2(FC) of more than 14 (Table 1).
Figure 2. Characteristics of lncRNA expression levels between H3N2- infected and non-infected groups. A Differentially expressed lncRNAs were analyzed by hierarchical clustering. B Volcano plot displaying differentially expressed lncRNAs in the two groups. C Distribution of differentially expressed lncRNAs in each chromosome.
LncRNA ID Locus Regulation P value Log2(FC) MSTRG.11930.8 15:71487937-71532738 Down 2.86E-30 -14.10977625 MSTRG.11930.3 15:71442625-71547216 Down 5.31E-09 -12.33057588 MSTRG.9036.1 12:130884797-130905045 Up 2.12E-24 12.07586697 MSTRG.18254.4 19:50095703-50115987 Up 2.64E-16 12.0778888 MSTRG.35702.1 9:111973032-111993029 Up 6.28E-24 12.11163237 MSTRG.18254.8 19:50101833-50115910 Up 5.45E-23 12.12016728 MSTRG.18254.11 19:50110235-50151173 Up 2.33E-68 12.21266154 MSTRG.18148.9 19:47920256-47947180 Up 7.01E-25 12.23237036 MSTRG.18148.7 19:47914962-47952571 Up 4.05E-82 12.3091667 MSTRG.18149.14 19:47927354-47944128 Up 1.4E-26 12.60624992 MSTRG.14746.1 17:31785546-31828849 Up 9.1E-87 12.62649567 MSTRG.18149.3 19:47911586-47949487 Up 2.18E-28 13.01068919 MSTRG.18149.4 19:47911586-47954970 Up 1.31E-28 13.07130734 MSTRG.18254.9 19:50104896-50151173 Up 2.39E-29 13.17550951 MSTRG.18254.7 19:50101833-50142753 Up 2.42E-29 13.51465658 MSTRG.15643.1 17:67298574-67317698 Up 2.16E-32 13.77495727 MSTRG.18254.13 19:50120946-50151173 Up 1.48E-40 15.22589906 MSTRG.18254.14 19:50126300-50151173 Up 1.49E-40 15.22595034 MSTRG.18254.15 19:50131635-50151173 Up 1.45E-40 15.22769837 MSTRG.18254.3 19:50095703-50137465 Up 5.14E-46 16.09175095 Table 1. The top 20 differentially expressed lncRNAs in H3N2-infected cells.
The differentially expressed lncRNAs were widely distributed in all chromosomes, although the numbers varied in different chromosomes. Most altered lncRNAs were located on chromosome 1, whereas few altered lncRNAs were located on chromosome Y (Fig. 2C).
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We also detected mRNA expression levels of A549 cells after H3N2 infection. Hierarchical clustering showed that the mRNA expression profiles were significantly altered in the H3N2-infected group compared with that in the noninfected group (Fig. 3A). In total, 50, 031 mRNA transcripts were found to be differentially expressed in A549 cells infected with H3N2 (FC ≥ 2, P ≤ 0.05) (Supplementary Table S4). Of the differentially expressed mRNA transcripts, 20, 907 were upregulated, and 29, 124 were downregulated (Fig. 3B). Moreover, 833 genes were upregulated with a FC of more than 100 after infection. In our study, the gene showing the greatest upregulation was RPL4, with a log2(FC) of 12.3, whereas the gene showing the greatest downregulation was EGFR, with a log2(FC) of -14.2 (Table 2).
Figure 3. Characteristics of mRNA expression levels between the H3N2-infected and non-infected groups. A Differentially expressed mRNAs were analyzed by hierarchical clustering. B Volcano plot displaying differentially expressed mRNAs in the two groups. C Distribution of differentially expressed mRNAs in each chromosome.
Gene symbol Ensembl ID Locus Regulation P value Log2(FC) EGFR ENST00000275493 7:55019101-55211628 Down 2.26E-31 - 14.2050352 AKR1C3 ENST00000605149 10:5077638-5107680 Down 5.19E-77 - 13.7632822 ACTN4 ENST00000440400 19:38724157-38731583 Down 9.31E-29 - 13.67219 PDXK ENST00000468090 21:43719097-43762307 Down 3.61E-28 - 13.6668779 DDX3X ENST00000457138 X:41333398-41350269 Down 3.37E-27 - 13.5112125 NCOR2 ENST00000429285 12:124324598-124535603 Down 5.66E-26 - 13.067891 VPS13C ENST00000395898 15:61867744-62060448 Down 8.61E-26 - 13.039242 SYNE2 ENST00000344113 14:63852983-64226433 Down 7.50E-26 - 13.038538 NFIC ENST00000589123 19:3359563-3469217 Down 1.46E-25 - 12.9738161 CD109 ENST00000287097 6:73696104-73828313 Down 1.50E-25 - 12.9729377 SCRN1 ENST00000426154 7:29920104-29990118 Down 4.07E-25 - 12.8686194 ITGAV ENST00000374907 2:186590080-186680897 Down 4.42E-25 - 12.8600254 ALDOA ENST00000338110 16:30053090-30070414 Down 1.97E-24 - 12.8504503 CANX ENST00000452673 5:179698906-179730925 Down 6.74E-25 - 12.8175625 PCDH9 ENST00000377861 13:67201015-67230445 Down 1.70E-24 - 12.7330165 ADD2 ENST00000264436 2:70656784-70768177 Down 1.63E-24 - 12.7275731 APP ENST00000354192 21:25880550-26170747 Down 7.25E-18 - 12.6405452 CIT ENST00000261833 12:119685791-119877288 Down 4.86E-24 - 12.6196307 PMEPA1 ENST00000265626 20:57648394-57711536 Down 7.01E-24 - 12.5766746 RPL4 ENST00000561775 15:66499346-66504827 Up 6.13E-24 12.03161073 Table 2. The top 20 differentially expressed mRNA in H3N2-infected cells.
Similar to the distribution pattern of lncRNAs, the differentially expressed genes in H3N2-infected A549 cells were unevenly distributed among chromosomes. Most altered mRNAs were found on chromosome 1, whereas few altered mRNAs were found on chromosome Y (Fig. 3C).
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Next, we systematically analyzed the basic features of the lncRNAs and compared them with protein-coding genes. The lengths of lncRNA transcripts were longer than those of mRNAs (Fig. 4A). Additionally, the number of exons of lncRNAs was lower than that of mRNAs (Fig. 4B).
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To better understand the roles of differentially expressed lncRNAs in H3N2-infected cells, GO and KEGG pathway analyses were used to explore the functions of cis- and trans- target genes of H3N2-induced lncRNAs. The results showed that the target genes of these lncRNAs were significantly enriched in biological processes, such as cellular metabolism. The top 20 significant GO terms are listed in Fig. 5A, 5B. The target genes of these differentially expressed lncRNAs participated in various signaling pathways, such as the B cell receptor signaling pathway and autophagy. The top 20 statistically significant enriched KEGG pathways are shown in Fig. 5C, 5D. These findings suggested that the induced lncRNAs regulated cellular metabolic processes, immunity, and autophagy during H3N2 infection. Because many genes were enriched in the cellular metabolic pathway, we further selected 10 of the most dysregulated lncRNAs to generate the cis- or transregulatory networks (shown in Fig. 5E, 5F).
Figure 5. GO enrichment and KEGG pathway analyses of target genes of H3N2-induced lncRNAs. A Top 20 significant GO biological process terms by cis-regulation. B Top 20 significant GO biological process terms by trans-regulation. C Top 20 significantly enriched pathways terms by cis-regulation. D Top 20 significantly enriched pathways terms by trans-regulation. E Cis-regulatory network of lncRNAs-mRNAs, F trans-regulatory network of lncRNAs/mRNAs. LncRNAs are represented as triangles, and mRNAs are represented as circles. Red nodes indicate upregulation, blue nodes indicate downregulation, and color shade indicates different degrees of dysregulation.
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Dysregulated mRNAs in H3N2-infected cells were used for GO enrichment and KEGG pathway analyses. As shown in Fig. 6A, the majority of the dysregulated mRNAs were significantly enriched in some biological processes, such as cellular metabolic process, organic cyclic compound metabolic process, and cellular macromolecule metabolic process, suggesting that H3N2 infection had a profound effect on cellular metabolism in A549 cells. Furthermore, KEGG functional analyses showed that dysregulated mRNAs were significantly enriched in some pathways (Fig. 6B), including citrate cycle (tricarboxylic acid [TCA] cycle), DNA replication, biosynthesis of unsaturated fatty acids, and autophagy. Taken together, these results suggested that the differentially expressed mRNAs belonged to multiple pathways.
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RT-qPCR was performed to further detect differentially expressed lncRNAs in sequencing data. Among the 6129 differentially expressed lncRNAs, we validated seven upregulated and two downregulated lncRNAs by RT-qPCR (Fig. 7A) and RT-PCR (Fig. 7B). The results showed that changes in lncRNAs expression levels were consistent with RNA-seq data. Moreover, we also tested the reported lncRNAs, and the results were consistent with the literature (Fig. 7B, 7C).
Figure 7. Validation of differentially expressed lncRNAs and reported lncRNAs by RT-qPCR and RT-PCR. p < 0.05 (*), < 0.01 (**), and < 0.001 (***) were considered statistically significant. A Validation of differentially expressed lncRNAs by RT-qPCR, B validation of differentially expressed lncRNAs and reported lncRNAs by RT-PCR and C validation of reported lncRNAs by RT-qPCR.