Differential expression of microRNA, miR-150 and enhancer of zeste homolog 2 (EZH2) in peripheral blood cells as early prognostic markers of severe forms of dengue

Abstract

Background: Dengue presents a wide clinical spectrum. Most patients recover following a self-limiting non-severe clinical course. A small proportion of patients progress to severe disease, mostly characterized by plasma leakage with or without hemorrhage. Early symptoms of severe dengue (SD) are similar to those of non-severe dengue fever (DF). Severe symptoms manifest after 3-5 days of fever, which can be life threatening due to lack of proper medications and inability to distinguish severe cases during the early stages. Early prediction of SD in patients with no warning signs who may later develop severe infection is very important for proper disease management to alleviate related complications and mortality. microRNA are small non-coding RNA molecules that regulate post-transcriptional gene expression. Due to the remarkable stability and the role of microRNA in gene expression, altered expression of microRNA was evaluated to explore clinically relevant prognostic markers of severe dengue.

Methods: The relative expression of microRNA hsa-let-7e (let-7e), hsa-miR-30b-5p (miR-30b), hsa-miR-30e-3p (miR-30e), hsa-miR-33a (miR-33a), and hsa-miR-150-5p (miR-150) and several putative target genes in peripheral blood cells (PBC) collected from 20 DF and 20 SD positive patients within 4 days from fever onset was evaluated by quantitative reverse transcription PCR (qRT-PCR).

Results: miR-150 showed significant (P < 0.01) up regulation in PBC of SD patients compared to DF patients during the acute phase of infection. Expression of enhancer of zeste homolog 2 (EZH2) was significantly (P < 0.01) down regulated indicating that genes involved in epigenetic regulation are also differentially expressed in SD patients during the early stage of infection.

Conclusions: Differential expression of microRNA miR-150 and the putative target gene EZH2 may serve as reliable biomarkers of disease severity during early stages of dengue infection.

Keywords: Acute dengue biomarkers; Dengue; Severe dengue; microRNA.

Fig. 1

Relative miR-150 expression in PBC samples in DF and SD patients. Relative expression at admission in patients recruited on, day 2 (n_DF = 2, n_SD = 3), day 3 (n_DF = 6, n_SD = 12), day 4 (n_DF = 11, n_SD = 5), all patients recruited within 3 days (n_DF = 8, n_SD = 15) and within 4 days (n_DF = 20, n_SD = 20) from fever onset. Relative expression presented as log values to the base 2 based on 2^−ΔCq values against geometric mean of miR-16 and miR-103a. * P < 0.01 based on ΔCq ± SEM using independent t – test with Bonferroni adjustment

Fig. 2

Relative EZH2 expression in PBC from DF and SD patients. Relative expression at admission in patients recruited on, day 2 (n_DF = 2, n_SD = 3), day 3 (n_DF = 6, n_SD = 12), day 4 (n_DF = 11, n_SD = 5), all patients recruited on day 3 & day 4 (n_DF = 17, n_SD = 17), within 3 days (n_DF = 8, n_SD = 15) and within 4 days (n_DF = 20, n_SD = 20) from fever onset presented as log values to the base 2 (log2) based on 2^−ΔCq values against GAPDH. * P < 0.01 based on ΔCq ± SEM using independent t – test with Bonferroni adjustment