Identification of miRNA biomarkers of pneumonia using RNA-sequencing and bioinformatics analysis

Abstract

Pneumonia is a lower respiratory tract infection that causes dramatic mortality worldwide. The present study aimed to investigate the pathogenesis of pneumonia and identify microRNA (miRNA) biomarkers as candidates for targeted therapy. RNA from the peripheral blood plasma of participants with pneumonia (severe, n=9; non-severe, n=9) and controls (n=9) was isolated and paired-end sequencing was performed on an Illumina HiSeq4000 system. Following the processing of raw reads, the sequences were aligned against the Genome Reference Consortium human genome assembly 38 reference genome using Bowtie2 software. Reads per kilobase of transcript per million mapped read values were obtained and the limma software package was used to identify differentially expressed miRNAs (DE-miRs). Then, DE-miR targets were predicted and subjected to enrichment analysis. In addition, a protein-protein interaction (PPI) network of the predicted targets was constructed. This analysis identified 11 key DE-miRs in pneumonia samples, including 6 upregulated miRNAs (including hsa-miR-34a and hsa-miR-455) and 5 downregulated miRNAs (including hsa-let-7f-1). All DE-miRs kept their upregulation/downregulation pattern in the control, non-severe pneumonia and severe pneumonia samples. Predicted target genes of DE-miRs in the subjects with non-severe pneumonia vs. the control and the subjects with severe pneumonia vs. the non-severe pneumonia group were markedly enriched in the adherens junction and Wnt signaling pathways. KALRN, Ras homolog family member A (RHOA), β-catenin (CTNNB1), RNA polymerase II subunit K (POLR2K) and amyloid precursor protein (APP) were determined to encode crucial proteins in the PPI network constructed. KALRN was predicted to be a target of hsa-mir-200b, while RHOA, CTNNB1, POLR2K and APP were predicted targets of hsa-let-7f-1. The results of the present study demonstrated that hsa-let-7f-1 may serve a role in the development of cancer and the Notch signaling pathway. Conversely, hsa-miR-455 may be an inhibitor of pneumonia pathogenesis. Furthermore, hsa-miR-200b might promote pneumonia via targeting KALRN.

Keywords: RNA-sequencing; miRNA; notch signaling pathway; pneumonia; protein-protein interaction; target.

Figure 1.

Venn analysis of DE-miRs from the comparison of the non-severe pneumonia and control groups, and the severe and non-severe pneumonia groups. Upward arrows in red represent upregulation and downward arrows in blue represent downregulation. DE-miRs, differentially-expressed microRNAs.

Figure 2.

Expression of 11 key DE-miRs in the plasma of the peripheral blood of the severe pneumonia, non-severe pneumonia and control groups. Key DE-miRs that are (A) upregulated and (B) downregulated. DE-miRs, differentially expressed microRNAs. RPKM, per kilobase per million reads.

Figure 3.

Heatmap from clustering analysis of DE-miRs in the plasma of the peripheral blood of the severe pneumonia, non-severe pneumonia and control groups. The x-axis denotes samples and the y-axis denotes miRNA. The Z-score indicates the parameter used for the calculation of miRNA expression based on raw reads. A value of >0 was deemed to indicate upregulation (red) and a value <0 was deemed to indicate downregulation (green). The purple, brown and blue panels at the top of the heat map indicate severe, non-severe and control samples, respectively. DE-miRs, differentially expressed microRNAs.

Figure 4.

Enrichment analysis of key DE-miR target genes from comparisons of (A) non-severe pneumonia and control groups, and (B) severe and non-severe pneumonia groups. DE-miR, differentially expressed microRNA. -log10(P-value) represents the significance of the enriched pathway and was used to quantify and present the results more clearly. P-values were calculated following pathway enrichment and P<0.05 was identified as the threshold for significant pathway selection.

Figure 5.

Enrichment analysis of the key DE-miRs. Names on the left are the pathway categories of the target genes. The circles of different sizes represent GeneRio, the ratio of the ‘number of target genes enriched in a specific pathway’ to the ‘number of total genes in the pathway’. Different colors represent the P-values of the significance of different pathways, compared with the reference species. The numbers in brackets underneath the miRNA names indicate the numbers of identified genes in each pathway category. DE-miRs, differentially expressed microRNAs.

Figure 6.

PPI network analysis of targets of the DE-miRs. Lines indicate interactions and the size of the circle indicates the number of interactions. PPI, protein-protein interaction; DE-miRs, differentially expressed microRNAs.