Identification of potential whole blood MicroRNA biomarkers for the blood stage of adult imported falciparum malaria through integrated mRNA and miRNA expression profiling

https://doi.org/10.1016/j.bbrc.2018.10.072Get rights and content

Highlights

  • A parallel microarray-based method studies host miRNA expression profile changes under malaria.

  • Most of putative pathways are involved in immune response processes during falciparum malaria.

  • Five miRNAs were identified as potential blood biomarkers for falciparum malaria.

Abstract

Correlating aberrant miRNA levels with imported malaria during the blood stage is required to better understand the in vivo biological and molecular processes that are involved in the response to Plasmodium falciparum infection and to find new biomarkers and diagnosis tools. We used a parallel microarray-based approach to investigate an integrated view of how the host miRNA expression profile changes in response to P. falciparum infection in whole blood obtained from six subjects with adult imported falciparum malaria (AIFM) and six normal subjects. We first investigated the functions of 1007 significantly differentially expressed genes from microarrays and predicted 56 putative pathways participating in malaria pathogenesis, which reflected systemic changes in the host under falciparum infection. Then, we validated the in silico-predicted targets of 50 differentially modulated miRNAs (7 upregulated and 43 downregulated) by examining the actual mRNA expression of these particular genes in our gene expression profile database; putative target gene sets were grouped into functional categories to investigate the roles of differentially expressed miRNAs. We considered 36 intersecting putative pathways, most of which were involved in multiple immune response processes, such as cell defense response, immune response, TNF signaling pathway, and T cell receptor signaling pathway, which may actually be regulated by differentially expressed miRNAs. Additionally, we identified five enriched upregulated miRNA sets (miR-3135b, miR-6780b-5p, miR-1246, miR-6126, and miR-3613-5p) as potential blood biomarkers of immunopathological status and prediction of early host responses, disease prognosis, and severe outcomes in AIFM.

Introduction

Malaria is one of the most important parasitic diseases of human beings and remains a major cause of morbidity and mortality both in tropical and subtropical regions [1]. According to the World Health Organization, an estimated 216 million cases of malaria occurred globally in 2016 leading to about 445,000 deaths [2]. More than 90% of global malaria mortality occurs in sub-Saharan Africa, mainly in children under five years old, caused by Plasmodium falciparum [1]. But, in China, the vast majority of falciparum malaria cases are in adult (93%) males (88%) and 90% of P. falciparum infections were imported into China from Africa [3]. There is a significant difference both in clinical and parasitological findings between adult semi-immune and non-immune patients, and the latter is a high-risk group for severe malaria [4]. Chinese non-immune adult male patients who go to malaria-endemic areas and get adult imported falciparum malaria (AIFM) have been observed to have an increased incidence of severe malaria and mortality. This indicates that the immune response would be different in this group under P. falciparum infection.

Malaria pathogenesis is due to a multigene consociation that involves a global change in gene expression profiling [5]. MicroRNAs (miRNAs) are a class of small non-coding RNAs principally involved in posttranscriptional silencing of mRNA [6]. Dysregulation of the expression of miRNAs is usually indicative of pathological disorders or physiological changes in the individual [6]. Though it is commonly recognized that P. falciparum does not have miRNA-sequences in its genome [7], host miRNAs might be reprogrammed to respond to Plasmodium infection and minimize disease severity [8]. Numerous studies have attempted to correlate aberrant host miRNA levels with malaria [9,10] and evaluate host miRNAs as promising biomarkers and diagnostic tools in different stages of the parasite's life cycle [9,11,12]. Likewise, many miRNAs are dysregulated in humans during different stages of malaria [[13], [14], [15]].

Understanding pathology and mortality, which from malaria are mainly caused during the blood stage of the parasite's life cycle [1], is crucial for the development of diagnosis and treatment strategies during pathogenesis of malaria. The parasite infection is controlled by cells, including the immune and nonimmune cells, existing in blood, through responding to parasite signals [16]. Additionally, blood is an accessible sample for capturing these effects in malaria patients. However, only one study has examined differential expression of 84 immune-related miRNAs in falciparum malaria whole blood. This study used a controlled human malaria infection to assess immune responses from subjects experimentally infected with P. falciparum following primary blood-stage malaria [17]. As the miRNA set is very limited, no holistic picture of host miRNAs activated during infection was obtained, preventing potential discoveries of novel genes and pathways with important roles during infection. In addition, no comprehensive data is available on expression profiles of miRNAs in whole blood during the blood-stage of AIFM.

Accumulating evidence has converged on recognizing that the onset of disease implicates complex biological processes. In this study, we attempted to correlate aberrant miRNA levels with AIFM during the blood stage in order to better understand in vivo pathogenesis and find new biomarkers and diagnostic tools. In order to do this, a systemic, integrated view of the host response to infection is required. We used a parallel microarray-based approach to investigate regulation following significant changes in the miRNA expression profile. We used Affymetrix miRNA 4.0 microarrays to examine the occurrence of miRNAs in peripheral whole blood specimens obtained from six subjects with AIFM and six normal subjects and validated their in silico-predicted targets through mRNA profiling using Affymetrix PrimeView microarrays in the same specimens.

Section snippets

Influence of infection on human whole blood mRNA expression profiling in the blood stage of AIFM

To identify genes that significantly contributed to the differences between infected patients and control individuals during the blood-stage of AIFM, we collected six whole blood specimens from adult male Chinese patients with AIFM who were originally infected in Angola before any treatment in June 2016. Six healthy subjects were used as a control. The characteristics of study subjects are listed in Table S1.

We performed an mRNA microarray (Affymetrix GeneChip PrimeView ™ Human Gene Expression

Discussion

Human immune defense against malaria parasites is highly complex, with numerous cells, genes, and pathways interacting and activating various components of the immune system [20]. Though the mechanisms by which malaria parasites stimulate host immune responses have been extensively reported [21], research about the molecular mechanisms of host immune responses to malaria infection in general is extremely challenging. Recently, miRNA microarray analysis approaches have helped many researchers

Acknowledgements

This work was supported by the Research Foundation of Beijing Municipal Commission of Health and Family Planning (Grant: 2015-3-010) and the Research Foundation of Capital Medical University (No.PYZ2017010), China.

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