Validation of the CV-B5 infection models
RD and SH-SY5Y cells infected with 1MOI CV-B5 at 24h can cause CPE, including rounding up, aggregation, and even death when compared with control cell (Fig. 1A). Western Bolt analysis also detected the viral specific protein VP1 in both RD and SH-SY5Y cells (Fig. 1B). Three independent experiments were carried out. These results showed that RD and SH-SH5Y cells were successfully permissive to CV-B5 infection.
Analysis and classification of differentially-expression of lncRNAs
To analyze the level of transcripts, RD and SH-SY5Y cells infected with CV-B5 were subjected to Human Genome Array. We obtained 6500 and 5375 novel lncRNAs in RD and SH-SY5Y cells respectively, based on CPC2, CNCI and PFAM analysis (Fig. 2A and 2B). With regard to the differentially-expressed genes, our transcript contained a total of 1268 lncRNAs in RD cell (Supplementary table 1) and 1603 lncRNAs in SH-SY5Y cell (Supplementary table 2). In total, 1264 lncRNAs with transcripts length ≥200 bp and multi-exon in nature were acquired from the primary lncRNAs in RD cell, of which, 46.2% were lincRNAs, 28.6% were anti-sense lncRNAs, 24.1% were sense overlapping lncRNAs, and 1.0% were sense intronic lncRNAs (Fig. 2C). On the other hand, 1598 lncRNAs were filtered from the primary lncRNAs in SH-SY5Y cell and they contained 48.6% lincRNAs, 34.7% anti-sense lncRNAs, 16.0% sense overlapping lncRNAs, and 0.8% sense intronic lncRNAs (Fig. 2D).
Using Cuffdiff and p<0.05 as the criteria, the differentially-expressed lncRNAs in RD and SH-SY5Y cells in response to CV-B5 infection were shown in clustering analysis (Fig. 3A). Preliminary microarray hybridization in RD cell identified 508 up-regulated and 760 down-regulated lncRNAs which greatly changed after CVB5 infection (Fig. 3B). And, 792 significantly increased and 811 significantly decreased lncRNAs were identified in SH-SY5Y cell infected with CV-B5 (Fig. 3C). Among them, some have been associated with neurological disorders, such as TCONS_00385873, TCONS_00087939 and TCONS_00450969, when compared to RD cell. Therefore, we conclude that these lncRNAs may have critical roles in the nervous system.
GO annotation and KEGG pathway analysis
We used the clusterProfiler to explore the gene ontology functional classes and pathways in order to further understand our data. The differentially-expression lncRNAs were used to conduct the GO enrichment and KEGG pathway analysis. The top 20 significant GO terms of molecular function, biological process, and cellular component, based on the p value and rich factors, were shown in Fig. 4. The results indicated that the most enriched GO terms in RD cell were single-organism process and membrane part, and cellular process (Fig. 4A). And cell part and protein binding were the most enriched GO terms in SH-SY5Y cell (Fig. 4B).
With regard to the KEGG pathway, the most enriched terms were mainly neuroactive ligand-receptor interaction, spliceosome, and viral myocarditis for CV-B5 infection in RD cell (Fig. 5A). On the other hand, the enriches terms in SH-SY5Y cell were mainly associated with several signaling pathways including ECM-receptor interaction, complement and coagulation cascades and TGF-beta signaling pathway (Fig. 5B).
RT-qPCR for validating the lncRNAs after CV-B5 infection
To further verify the accuracy of the RNA sequencing analysis, the significantly up- and down- regulated lncRNAs were selected and validated using RT-qPCR. Seven lncRNAs were selected for further validation. Fold changes in the CV-B5 infected cells were calculated using the 2-△△CT method (three independent experiments). Results showed that three lncRNAs were up-regulated and four lncRNAs were down-regulated (Fig. 6). These findings were in accordance with our data analysis, which indicated that fold change results determined by RT-qPCR were lower than those determined using RNA sequencing results.
The analysis of antisense lncRNA-IL12A
The function of lncRNAs needs further experimentation because it cannot only be inferred from the RNA sequencing results. LncRNA-IL12A belonged to antisense lncRNA which related the gene Interleukin-12 subunit alpha and located on chromosome 3. In our present study, we selected lncRNA-IL12A in order to illustrate its roles in CV-B5 replication. Firstly, we predicted the secondary structure of lncRNA-IL12A (Fig. 7A). Then we contrasted pcDNA3.1-2Flag-lncRNA-IL12A transfect into 293T and found it does not have the ability to code (Fig. 7B). Next, we overexpressed and silenced its RNA expression in order to determine CV-B5 VP1 expression and our results showed that LncRNA-IL12A inhibits viral replication (Fig. 7CD). Finally, from the KEGG analysis we found the lncRNA-IL12A enriched in the Wnt signaling pathway and the key molecules downstream (C-myc and CyclinD1) siginicantly changes by real-time RT-PCR (Fig. 7E).