Currently, miRBase [25, 26] version 22.1 (accessed on 08/02/2021) reports 1917 entries for stem-loop miRNA on the human genome resulting in 2654 mature miRNAs. On HSA21, there are 30 precursors reported, resulting in 38 mature miRNAs. Of these precursors, only five HSA21 sequences are classified as “high confidence miRNAs” (i.e. present high levels of certainty that they do exist) or present relevant deep sequencing data (i.e. mean of counts reads per million) (Online Resource 7). The miRNAs miR-155, miR-802, miR-125b-2, let-7c, and miR-99a, located on HSA21, have been the most extensively studied and implicated in the DS pathogenesis [27,28,29,30,31,32]. These miRNAs present two mature forms (-5p and -3p), except miR-802 [25]. In the present study, we investigated the expression pattern of all -5p and -3p mature forms of miR-155, miR-125b-5p, and miR-99a, besides miR-802; however, only -5p mature forms were detected in our samples.

The four HSA21 mature miRNAs detected in the present study did not present significant differential expression patterns between children with DS and controls after FDR adjustment. Although the overexpression of HSA21-located miRNAs has been associated with DS phenotypes [27], including low blood pressure [28], dementia [29], and altered immune processes [30, 31], there are few studies concerning miRNA expression in blood cells from individuals with DS. Xu et al. [33] identified overexpression of miR-99a, let-7c, miR-125b-2, and miR-155 in lymphocytes from children with DS by high-throughput sequencing technology and validated their data using quantitative RT-PCR. Another similar study by the same group [34] showed the same miRNAs downregulated in fetal cord blood mononuclear cells from fetuses with DS. On the other hand, comparable expression of the miRNAs miR-99a, let-7c, and miR-125b-2 were observed between cultured PBMCs from children with DS and age-matched healthy donors while overexpression of the miR-155 was observed in DS cells compared with control ones, investigated by quantitative RT-PCR [31]. Considering the different study designs, in which different methodologies for quantifying miRNAs and analyzing results were used, additional studies are necessary to better elucidate the expression pattern of HSA21 miRNAs and its consequence for the DS phenotype.

The six DEmiRs identified in the present study between children with DS and controls are not located on HSA21; four of them were observed to be upregulated (miR-378a-3p, miR-130b-5p, miR-942-5p, and miR-424-3p) and two downregulated (miR-452-5p and miR-668-3p). Although some DEmiRs identified in the present study have been previously found to be dysregulated in other DS tissues [35,36,37], to our knowledge, this is the first study to report altered expression of these six DEmiRs in PBMCs from individuals with DS. Altered expression of miRNAs not located on HSA21 with possible involvement in DS phenotypes has been previously reported [33,34,35,36,37,38,39,40], supporting the existing hypothesis of secondary transcriptional changes as a consequence of the trisomy 21 [41].

According to the literature, sex can influence gene expression levels [42, 43], as well as the expression of miRNAs [44]. Considering that in the present study the sex was not matched between individuals with DS and controls, we investigated if there was difference in the expression of the six DEmiRs and the four HSA21 mature miRNAs between male and female participants regardless the study group (case or control). miR-452-5p and miR-668 were observed to be overexpressed in females (data not shown), indicating a possible sex bias in miRNA expression. Therefore, we compared the expression of these two miRNAs between case and control groups considering only female participants (data not shown) and obtained the same results from the total sample (both downregulated in the DS group), indicating that there is no sex bias in our results. Data on sex and age for each sample include in the study are available in the Online Resource 1.

Enrichment analyses of GO biological processes and KEGG pathways including the 2401 non-redundant predicted targets of the six DEmiRs point to the contribution of these genes to pathways related to the immune system, including the TLRs, TGF-β, and Hippo signaling pathways. TLRs are key elements of innate immunity as they participate in the recognition of pathogens, antigen presentation, apoptosis, and production of interferons by the virus-infected cells [45]. TLR signaling is dysregulated in children with T21 and this impairment is believed to contribute to the chronic inflammation and sepsis observed in DS [46]. The TGF-β signaling pathway is a pivotal regulator of immune responses, participating in the proliferation, survival, activation, differentiation, and repertoire diversity of B- and T- cells under normal conditions and immune challenges [47]. Moreover, TGF-β regulates the development and functionality of innate cells (dendritic cells, macrophages, granulocytes, and natural killer cells) and the peripheral tolerance against self-antigens [47]. In DS, increased TGF-β levels have been previously associated with transient abnormal myelopoiesis and complications of this condition [48] and pointed as a candidate biomarker for prenatal diagnosis [49].

Studies have revealed extensive roles for the Hippo signaling pathway in adaptive and innate immune systems regulation. Components of the Hippo pathway (MST1/2) are involved in T-cell development and differentiation, B cell homeostasis, and they play key roles in antigen-presenting cells, such as macrophages and dendritic cells [50]. Also, there is evidence of crosstalk between Hippo and TLR signaling pathways [51], along with other signaling networks involved in immune regulation [52]. To our knowledge, there are no studies concerning the role of the Hippo pathway in the DS phenotype and our study is the first to point out a possible association between Hippo pathway dysregulation and DS.

Several of the predicted HSA21-located target genes of our DEmiRs play a role in the immune system. BACH1 is involved in the regulation of the expression of macrophage-associated genes, B-cell development, and antigen presentation [53]; BRWD1 cooperates with networks to drive B-cell development [54]; ERG participates in the control of B lymphopoiesis [55]; GABPA is a regulator of cytokine secretion [56]; RUNX1 regulates TLR signaling pathways and inflammatory cytokine production [57]. Besides the fact that they are present in triplicate in individuals with DS, the altered expression of miRNAs that target these genes could be modulating their expression pattern giving further support to the role of these miRNAs in DS pathogenesis.

Previous studies from our research group showed dysregulation of immune and inflammation-related genes non-located on HSA21 in children with DS [13, 58]. The study of immune-related genes [13] was carried out in the same cohort of individuals with DS and controls included in the present study; thus, we deemed it interesting to investigate a possible interaction between the immune-related DEGs previously identified and the six DEmiRs identified in the present study. Fourteen possible miRNA–target interactions were found (Table 3). These immune-related genes are involved in key immune response processes, such as regulation of T and B cells, production of cytokines, antibodies and autoantibodies, activation of signaling pathways, and their dysregulation could negatively influence the immune response in DS, as previously discussed [13].

Immune dysregulation has been widely described in DS [6, 59,60,61]. In line with the contribution of immunological alteration and infectious diseases to the pathophysiology of DS, individuals with T21 present increased morbidity and mortality due to respiratory tract infections [62, 63]. Since the start of the COVID-19 pandemic caused by SARS-CoV-2, there is concern about the individuals with DS being a risk group for the disease [64]. In fact, studies have shown that they present an increased risk for severe disease course and COVID-19–related hospitalization and death [65,66,67]. Recent network analysis of DS and SARS-CoV-2 revealed that the susceptibility to infection, worse prognosis, and complications of COVID-19 could be related to genetic factors associated with DS that predispose to unfavorable conditions, such as the dysregulation of genes involved in the immune response [68].

Our findings suggest that altered miRNAs expression patterns could be contributing to the immunological dysfunction observed in children with DS by targeting genes involved in several key processes crucial for the immune system to function properly. We emphasize the importance of investigating and understanding the immune system of DS individuals who constitute a clinically vulnerable population so that they can have better support and clinical management, and consequently  be better  in a future pandemic scenario. Considering that one coding gene can be regulated by multiple miRNAs [69] and one miRNA can regulate multiple targets [70], studies performing high throughput screening of miRNA and mRNA expression patterns could contribute to the understanding of the gene expression regulation in DS. Possible miRNA–target interactions identified in the present study are candidates for further investigation and experimental validation to understand the impact of DEmiRs on the mRNA expression of specific genes and consequently on the pathophysiology of DS.