miRNome analysis reveals mir-155-5p as a protective factor to dengue infection in a resistant Thai cohort

Abstract

Dengue virus (DENV) is a global health threat, with approximately 390 million infections annually, ranging from mild dengue fever to severe dengue hemorrhagic fever and shock syndrome. MicroRNA (miRNA) are crucial post-transcriptional regulators which may regulate host resistance to DENV infection. This study aimed to identify miRNAs involved in natural resistance to DENV infection. Individuals from a dengue-endemic area were classified as susceptible (SD) or resistant (RD) according to their anti-DENV antibody status. RD individuals were seronegative despite high local DENV infection prevalence. Monocytes susceptibility to DENV infection was assessed in vitro. The miRNome profiles of the monocytes from 7 individuals per group were assessed upon mock or DENV-2 infection. The antiviral effect of differentially expressed miRNAs was analyzed using miRNA mimics in HeLa cells followed by infection with DENV-1, DENV-2, DENV-3, and DENV-4 serotypes. We performed RNA-seq on miRNA mimic-transfected cells to identify miRNA-targeted genes interacting with DENV proteins. Monocytes from RD individuals exhibit lower DENV-2 production in vitro. The miRNAs miR-155, miR-132-3p, miR-576-5p were overexpressed in monocytes from RD group upon DENV-2 infection. The transfection of miR-155-5p mimic reduced DENV infection and viral production in HeLa cells, regulating 18 genes interacting with DENV proteins and downregulating target genes involved in interferon response, TP53 regulation, apoptosis, and vesicle trafficking (e.g. HSD17B12, ANXA2). Therefore, we show that monocytes from RD individuals show a distinct miRNA expression profile and reduced viral production. In vitro miR-155-5p upregulation induces an antiviral state, revealing potential therapeutic targets to treat dengue.

Keywords: Dengue; RNA-Seq; Resistance; miR-155-5p; miRNome; microRNA.

Fig. 1

Phenotypic/immunological data of individuals, and viral titration of their monocytes after DENV-2 infection. (A) Gender, age and DENV antibodies detection by hemagglutination inhibition in the 2 groups. (B) Log10 viral titer median on days 1, 2, 3, 4, 5 post infection. Error bar figures inter-quartile range. Statistics (Mann-Whitney test) shows significant difference on days 1, 2 and 3 with p-value = 0.0006 (***) but no significant difference on days 4, 5

Fig. 2

Representation of differentially expressed miRNA. (A to C): Volcano plots showing changes in miRNA expression levels according groups (RD = Resistant to dengue viral infection, SD = Susceptible to dengue viral infection). Red dots denote significantly differentially expressed miRNA (q-value < 0.05, Log10 q-value > 1.3) whose expression changed by more than 1.25-fold. Vertical line represents log2FC ± 0.32 threshold, and horizontal dotted line represents statistical significance threshold (q-value = 0.05). (D) Overall view of expression of selected miRNA. Salmon boxes represent overexpression while blue boxes mean reduction of expression. In the center of each colored box is the Fold Change (+/-) N.S. Not significant: q-value ;> 0.05

Fig. 3

Functional study to show effect of miRNA mimic transfection on DENV infection. (A) Cytometry on DENV-1 infected HeLa cells, 48 h post infection, transfection 24 h, and (B) viral titration of supernatants of those cells. (C) Cytometry on DENV-1, DENV-2, DENV-3, DENV-4 infected HeLa cells, 48 h post infection, transfection 24 h. (D) Viral titration of supernatants of those cells. Statistics: Mann-Whitney non-parametric tests ** p = 0,0079; * p = 0,0159

Fig. 4

RNA-Seq in HeLa cells transfected with miR-155-5p mimic or scrambled mimic. (A) Venn diagram representing DEG in Hela cell transfection (pink), DENV-Human protein interactions registered in DENVHunt Database (grey) and experimentally validated targets of miR-155-5p from miRTarbase and Tarbase (yellow). (B) Bubble plot representing biological terms from Reactome enriched with the 260 DEGs targeted by miR-155-5p as registered in miRTarbase and Tarbase. The 30 terms with lower FDR are shown. Bubble size refers to the number of genes that compose the biological term. Chromatic scale indicates the percentage of genes that are DEG in each biological term. Transcript. = Transcription. (C) Heatmap of the 18 DEG in Hela cell transfection experiment defined as experimentally validated targets of miR-155-5p and that interact with DENV proteins. Chromatic scale represents Z-score of normalized counts