Clinical characteristics of women participants
HM derived exosomes were isolated from both HIV-1 infected and uninfected control women and processed for miRNA profiling. A schematic illustration of the study layout is shown in Figure 1. In this study cohort, both HIV-1 infected and uninfected women were recruited from the Plateau State, Nigeria as described [20, 28]. Although, a series of HM samples were collected and shipped to Canada, a total of 36 HM samples from the first week postpartum (27 HIV-1 positive and 9 HIV-1 negative as controls) were processed for HM exosome miRNA profiling. The details of the women (average age 31 years) who participated in the study are shown in Table 1 which revealed that out of 27 HIV-1 positive women, 22 were carrying HIV-1 for 4~15 years whereas 5 women were carrying HIV-1 for only 3 years. Further, all HIV-1 positive women were on ART (CD4+ count ≤ 300/mm3 with undetectable viral load) and receiving antiretroviral drugs according to the regimen set by Nigerian Government and the WHO as described [29]. It was impossible to obtain samples from ART-naïve HIV-1 positive women. Number of years on ART were counted, the day a woman was diagnosed positive for HIV-1 and placed on ART. As shown in Table 1, 13 out of 22 HIV-1 positive women showed high infant mortality compared to the control women whereas 5 HIV-1 positive women never had experienced any pregnancy. In addition, upon follow-up infants born to these HIV-1 positive women were found stunted and underweight as described [18].
Milk exosome characterization and RNA quality check
HM samples were processed for exosome isolation and confirmed by TEM. A representative image of HM exosomes is shown in Figure 2A which revealed that HM derived exosomes are 30-100 nm in size and are largely spherical in shape. For further confirmation of exosomes, a Western blot analysis was performed using an exosome protein marker CD81. Different concentrations of exosome proteins extracted from either freshly isolated or exosomes kept at room temperature for two days were loaded which confirmed the presence and stability of exosomes in HM (Figure 2B). Next, exosomes were processed for RNA isolation. RNAs were isolated from all HIV-1 positive and negative HM samples with an average yield of 40 ng/µl as quantified by Nanodrop spectrophotometer. The isolated total RNAs showed spikes distinctive of noncoding RNA bands at >25 nucleotides using as Agilent 2100 Bioanalyzer (Figure 2C & 2D). RNA samples which did not pass the quality check were excluded and replaced with new RNA samples, thus, only high quality RNAs were processed for miRNA NanoString profiling.
Identification of HM derived exosomal miRNAs from HIV-1 infected women
Differentially expressed exosomal miRNAs from HM samples obtained from both HIV-1 positive and negative women were profiled using the Human nCounter miRNA ver 3.0 which interrogates 800 human miRNAs (NanoString). A total of three cartridge chips were run at the same time each consisting of 12 samples (9 HIV-1 positive and 3 negative control per chip). Differentially expressed miRNAs were identified between HIV-1 positive and negative control women using log base 2-fold change (FC) of the levels of miRNAs between HIV-1 positive group and non-HIV-1 control group. A volcano plot showing miRNAs with altered expression in HIV-1 infected HM samples is shown in Figure 3. A total of 41 miRNAs were found to be significant (p <0.05) in HIV-1 positive women compared to control women. A complete list of all 41 miRNAs is shown in Suppl Table 1. As shown in Table 2, out of 19 miRNAs, which were differentially regulated (p <0.05; FC >1.3), 13 were found upregulated (hsa-miR-320e; hsa-miR-630; hsa-miR-148a-3p; hsa-miR-23a-3p; hsa-miR-378g; hsa-miR-30a-5p; hsa-miR-93-5p; hsa-miR-497-5p; hsa-miR-200b-3p; hsa-miR-200a-3p; hsa-miR-16-5p; hsa-miR-1262 and hsa-miR-4516) and 6 were found downregulated (hsa-miR-422a; hsa-miR-644a; hsa-miR-520a-5p; hsa-miR-506-5p; hsa-miR-1257 and hsa-miR-1253). From hereon, the prefix hsa was removed from the miRNAs.
Top ten miRNAs and their potential target genes
Since miRNAs act by directly silencing and reducing the expression of their target genes, we next predicted the potential targets of the top ten differentially expressed miRNAs using Tarbase v8.0 which produces a list of validated target genes [25]. As a result, we obtained a total of 19895 interactions. Some of the target genes are shown in Table 3. miR-16-5p was found to target the largest number of genes including IRF9, TLR4, TLR6, JUN followed by miR-30a-5p, miR-93-5p and miR-200b-3p (Table 3). miR-630 was shown to target BCL2, BCL2L11 and YAP1. miR-378g was shown to target 123 genes including SMAD2, CREBBP, WDR5 etc. Interestingly, multiple miRNAs were found to be involved in targeting a single mRNA gene such as BCL2L11 that was a target of miR-320e and miR-148a-3p. BCL2L2 was a target of miR-630 and miR-497-5p. NOTCH1 was a target of miR-30a-5p and miR-200b-3p. SMAD2 was a target of miR-378g, miR-497-5p and miR-16-5p (Table 3).
GO and KEGG pathways
KEGG pathway analysis on the predicted targets using DIANA-mirPath v 3.0 led to the identification of 31 significant KEGG pathways in which the predicted miRNA targets were enriched (Figure 4). A heatmap was generated from DIANA-miRPath v3.0, which shows miRNA targets associated with HIV-1 and belonged to multiple pathways such as Pathways in Cancer, Viral carcinogenesis, Adherens Junctions, TGF-β, Fatty acid Biosynthesis, p53 signaling, Cell Cycle, Pathways regulating pluripotency of stem cells, Proteoglycans in Cancer etc (Figure 4). Next, we performed GO analysis to identify the biological processes associated with the miRNAs. A total of 30 GO biological processes were observed (Figure 5). The highest enrichment GO terms targeted by these miRNAs included Biosynthetic Process followed by Viral process, Catabolic process, Cell death, Ion binding, Membrane organization, Mitotic cell cycle, RNA metabolic process, poly (A) RNA binding, Neurotropin TRK receptor signaling pathway etc (Fig 5).
miRNA-Gene interaction network
To understand the association of differentially expressed miRNAs in HIV-1 infected HM and their target proteins, miRNA-gene interaction network was generated using miRNet tool. The 19 differentially expressed miRNAs were uploaded into miRNet platform and miRNA-gene interactions were observed which generated 4190 target nodes and 6042 edges. Shortest Path filter with “All but miRNA nodes” generated 124 nodes with 105 targets and 393 edges (Figure 6). The top cluster hubs were miR-16-5p followed by miR-497, miR-93-5p, miR-30a-5p and miR-23a-5p. The biological functions were determined within the “reactome database” using the “hypergeometric test” algorithm and p-value <0.05. Results showed that Pre-Notch transcription and translation (TP53, E2F3, AGO2, CCND1) was the top group followed by Mitotic G1/S phase (Wee1, CDK6, CDKN1A, E2F3, PSMD11, CCND1, CCNE2), Cyclin D associated events in G1 (E2F3, CDK6, CDKN1A, CCND1), EGFR signaling (EGFR, GRB2, HSP90AA1), Intrinsic pathway of apoptosis (BCL2, XIAP, TP53, DYNLL2) and Cell cycle (BIRC5, CCND1, CDK6, CDKN1A, CCNE2, WEE1, TP53, PSMD11, PAFAH1B1, HSP90AA1, E2F3, TAOK1, FOXM1, CSNK2A1). Collectively, these results demonstrate that the target genes of miRNAs perturbed by HIV-1 belong to multiple biological pathways.
ROC curves
In order to evaluate the utility of exosomal miRNAs in HM, we performed ROC curve analysis of top 5 miRNAs in discriminating HIV-1 infected women from healthy controls which showed that miR-320e, miR148a-3p, miR-378g, miR-630 and miR-23a-3p have ROC AUC values of 0.75 (95% CI= 0.58 to 0.75), 0.79 (95% CI= 0.6 to 0.75), 0.83 (95% CI= 0.67 to 0.83), 0.82 (95% CI= 0.67 to 0.82) and 0.72 (95% CI= 0.55 to 0.72) respectively (Figure 7). Further, when miR-630 and miR-378g were combined, it yielded ROC AUC of 0.86 (95% CI= 0.72 to 0.86) (Figure 7) thus suggesting that miR-630 and miR-378g could serve as biomarkers to distinguish HIV-1 infected women from healthy controls and could accurately discern HIV-1 from control.
Hypothetical model showing miR-378g mediated HIV-1 transactivator (TAR) binding protein 2 (TARBP2) depletion and its predicted role in HIV-1 infection
TargetScan predicts the targets of miRNAs by searching for the presence of conserved 8-mer, 7-mer and 6-mer sites that match the seed regions of each mRNA [13]. TargetScan ver 7.2 database was utilized to predict and identify the target genes of miR-378g. In order to identify which of these potential targets are relevant to HIV-1, TargetScan results were interrogated with the search term HIV. Results showed that miR-378g has one target site in 3’UTR of TARBP2 (ENST 00000552857.1) from 382-388 (Figure 8A). TARBP2 is known to promote HIV-1 LTR expression and viral production whereas its siRNA-mediated knockdown inhibits HIV-1 LTR expression and viral production [30]. A schematic of the hypothetical layout is shown in Figure 8B, where we speculate that miR-378g mediated RNA interference would lower HIV-1 expression and viral production essentially as previously described [31].