Differential expression of miRNAs in enterovirus 71-infected cells - PubMed (original) (raw)

Differential expression of miRNAs in enterovirus 71-infected cells

Meng Xun et al. Virol J. 2015.

Abstract

Background: Enterovirus 71 (EV71) is one of the major etiological pathogens of hand, foot and mouth disease (HFMD) and can cause severe cerebral and pulmonary complications and even fatality. MicroRNAs (miRNAs), a class of small non-coding RNA molecules, play an important role in post-transcriptional regulation of gene expression and thereby influencing various physiological and pathological processes. Increasing evidence suggests that miRNAs act as key effector molecules in the complicated pathogen-host interactions. However, the roles of miRNAs in EV71 infection and pathogenesis are not well understood.

Methods: To identify special miRNAs involved in EV71 infection, a microarray assay was performed to study the expression pattern of miRNAs in EV71-infected human rhabdomyosarcoma cells (RD cells) and uninfected RD cells. We further predicted the putative target genes for the dysregulated miRNAs using the online bioinformatic algorithms (TargetScan, miRanda and PicTar) and carried out functional annotation including GO enrichment and KEGG pathway analysis for miRNA predicted targets. Then, the results of microarray were further confirmed by quantitative RT-PCR.

Results: Totally, 45 differentially expressed miRNAs ware identified by microarray, among which 36 miRNAs were up-regulated and 9 were down-regulated. 7166 predicted target genes for the dysregulated miRNAs were revealed by using TargetScan in conjunction with miRanda and PicTar. The GO annotation suggested that predicted targets of miRNAs were enriched into the category of signal transduction, regulation of transcription, metabolic process, protein phosphorylation, apoptotic process and immune response. KEGG pathway analysis suggested that these predicted target genes were involved in many important pathways, mainly including endocytosis and focal adhesion, MAPK signaling pathway, hypertrophic cardiomyopathy, melanogenesis and ErbB signaling pathway. The expression levels of 8 most differentially up-regulated miRNAs and 3 most differentially down-regulated miRNAs were confirmed by qRT-PCR. The expressions of hsa-miR-4530, hsa-miR-4492, hsa-miR-6125, hsa-miR-494-3p, hsa-miR-638, hsa-miR-6743-5p, hsa-miR-4459 and hsa-miR-4443 detected by qRT-PCR were consistent with the microarray data.

Conclusion: These results might extend our understanding to the regulatory mechanism of miRNAs underlying the pathogenesis of EV71 infection, thus strengthening the preventative and therapeutic strategies of HFMD caused by EV71.

PubMed Disclaimer

Figures

Figure 1

Figure 1

The morphological changes of EV71-induced cytopathic effect in RD cells were observed under a light microscope at 20× magnification at 48 h p.i.. RD cells were infected with EV71 at a m.o.i. of 0.1. (A) EV71-infected RD cells exhibited severe CPE appearance in response to virus replication at 48 h p.i..; (B) Uninfected RD cells showed normal morphology.

Figure 2

Figure 2

Heat map and unsupervised hierarchical cluster analysis of the differentially expressed miRNAs in EV71-infected cells and control cells. Total RNA from EV71-infected RD cells and uninfected RD cells in triplicate was used for microarray. Differentially expressed miRNAs were chosen with a log2 Ratio ≥ 0.5 and ≤ −0.5 and an adjusted P value < 0.05. Then columns and rows represent samples and particular miRNAs. The samples were correctly grouped together into EV71-infected cells and control cells according to the expression pattern The miRNA clustering tree is shown on the left. The color scale illustrates the relative expression level of miRNAs. Red color represents that the miRNA has higher expression in EV71-infected cells than in control, while green color represents that the miRNA has lower expression in EV71-infected cells than in control. The codes on the legend are log2-transformed values.

Figure 3

Figure 3

GO categories and distribution for the predicted miRNA targets related with EV71 infeciton. The GO terms were sorted by the number of genes in an ascending order from top to bottom. Biological process is enriched in signaling transduction, regulation of transcription, multicellular organismal development, cell adhesion and ion transport. Cellular components are enriched in nucleus, cytoplasm and membrane. Molecular function is enriched in protein binding, metal ion binding and zinc ion binding.

Figure 4

Figure 4

Validation of the differential expression of 11 miRNAs identified in the microarray by qRT-PCR. U6 was used as a reference gene. The expression levels of miRNAs in uninfected RD cells were set as 1. The expression levels of most miRNAs detected by qRT-PCR were correlated with those detected by microarray. (A) 8 up-regulated miRNAs (hsa-miR-4530, hsa-miR-4492, hsa-miR-4505, hsa-miR-6125, hsa-miR-494-3p, hsa-miR-638, hsa-miR-6743-5p, hsa-miR-4459). (B) 3 down-regulated miRNAs (hsa-miR-29a-3p, hsa-miR-4443, hsa-miR-27b-5p). *P < 0.05.

References

    1. Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH. Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis. 2010;10:778–90. doi: 10.1016/S1473-3099(10)70194-8. -DOI -PubMed
    1. Yi L, Lu J, Kung HF, He ML. The virology and developments toward control of human enterovirus 71. Crit Rev Microbiol. 2011;37:313–27. doi: 10.3109/1040841X.2011.580723. -DOI -PubMed
    1. Zhu FC, Liang ZL, Li XL, Ge HM, Meng FY, Mao QY, et al. Immunogenicity and safety of an enterovirus 71 vaccine in healthy Chinese children and infants: a randomised, double-blind, placebo-controlled phase 2 clinical trial. Lancet. 2013;381:1037–45. doi: 10.1016/S0140-6736(12)61764-4. -DOI -PubMed
    1. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116:281–97. doi: 10.1016/S0092-8674(04)00045-5. -DOI -PubMed
    1. Lee Y, Ahn C, Han J, Choi H, Kim J, Yim J, et al. The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003;425:415–9. doi: 10.1038/nature01957. -DOI -PubMed

MeSH terms

Substances

LinkOut - more resources