3.1 Gene Ontology and KEGG Analysis
miRNAs have been mainly studied in processes such as cancer and cardiovascular events. In contrast, the miRNA profile and its effects in response to probiotics and other microbiota microorganisms are still scarce [12]. Previously, in our workgroup, the miRNA miR-671-5p was upregulated in porcine monocytes stimulated with the probiotic BB12 [(NCBI GEO dataset (GSE132995)] and validated in moDCs [6]. In the present work, we performed functional analysis focused on immune system processes from a list of genes predicted to be targeted by miR-671-5p. The study resulted in 14,841 genes selected with a value of minimal free energy (mfe) ≤-25 kcal/mol and included only the best target per gene. Then, Gene Ontology (GO) enrichment analysis was performed to analyze the function of immune processes of the target genes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated which of the predicted target genes were mainly enriched in pathways. The ClueGo network of terms and pathways [13] is presented in (Fig. 1). The network contains 48 terms in 23 groups of immune system processes and pathways involved with miR-671-5p. Among them, negative regulation of the inflammatory response to an antigenic stimulus, focal adhesion, cell adhesion molecules, defense response to bacteria, immune response-activated cell surface receptor signaling pathway, and MAPK signaling pathway are directly related to the modulation of the immune response associated with symbiosis of gut microbiota and the host [4, 14].
Other functions and pathways are involved in cancer (microRNAs in cancer, pathways in cancer, small cell lung cancer), and there is a recent association between miR-671-5p in human cancers. The decrease in miR-671-5p expression is linked with metastasis and a poor prognosis in breast cancer [15]. Conversely, the overexpression of miR-671-5p is associated with the acceleration of proliferation, migration, and invasion of colon cancer cells [16] and prostate cancer [17]. As such, miR-671-5p has been proposed as a possible colon cancer biomarker. Findings on the expression of miR-671-5p in human cancers are contradictory. They should not be compared since they are tissue dependent, with various factors involving the microenvironment, hormones, cytokines, and activation of immune receptors, among others [18].
3.2 Transient downregulation of miR-671-5p
Previously, according to the in silico prediction, the mRNA of IL-10 could be a target of miR-671-5p (minimal free energy= -41.2 kcal/mol), and a modest decrease in IL-10 was found in moDCs stimulated for six hours with the probiotic BB12 [6]. Then, to investigate whether miR-671-5p downregulates the IL-10 transcript after BB12 stimuli in moDCs, transient silencing of miR-671-5p was carried out using porcine moDCs [20, 21]. Flow cytometry was employed to determine the transfection efficiency. 5'FAM-SCR (a fluorescent scramble sequence) was internalized in the cells using a cationic lipid (Lipofectamine®-RNAiMAX). The flow cytometry strategy used was previously reported by Rodríguez-Gómez et al. [19]; first, moDCs according to the size and complexity parameters (Fig. 2A). Then, myeloid cells (CD172a+) expressed 5'FAM-SCR (Fig. 2B). These cells were later analyzed for the expression of CD172a+ and SLA-DR+, a marker of mature immune presenting cells (Fig. 2C). The transfection efficiency was 46.3±28.4% (mean ± SD), similar to what has been observed in cell lines using the same transfection system [22, 23]. This decrease was sufficient to downregulate miR-671-5p expression in the Lipo+miR-671-5p-ASO+BB12 moDC group in comparison with the BB12-stimulated moDC group (Fig. 2D). All experiments were performed after five days of differentiation plus 48 h of transfection, followed by four h of stimulation with BB12 if it corresponded (n=3).
3.3. Quantification of IL-10
Subsequently, we expected that miR-671-5p downregulation would increase IL-10 transcripts. Indeed, the results indicate that the downregulation of miR-671-5p increases the mRNA expression of IL-10 (Fig. 3A). Then, the production of IL-10 cytokines was determined in the following groups of moDCs: Control, BB12-stimulated, Lipo+miR-671-5p-ASO, Lipo+miR-671-5p-ASO+BB12. Given that the probiotic BB12 is mainly recognized by Toll-like receptor 2 (TLR2) expressed by antigen-presenting cells, another group of moDCs was stimulated with the synthetic ligand of TLR2 Pam3CSK4 (miR-671-5p-ASO-Pam3CSK4). All groups produced IL-10 cytokines, but no significant differences were found (Fig. 3B). Despite IL-10 cytokine showing a tendency to increase in all the transfected groups of moDCs, it was not remarkable, probably because 4 h of stimuli with the probiotic was not sufficient for that aim. IL-10 is an autocrine cytokine, and an increase in this cytokine was previously found at 24 h of stimulation with BB12 [6, 24], and it was TLR2-dependent in porcine monocytes stimulated with BB12. These findings indicate that miR-671-5p may participate in the downregulation of IL-10 transcripts during the first four hours of stimulation with probiotic BB12.
Given that pigs are considered a better model to study human microbiota, future in vivo studies could help examine the participation of miR-671-5p and others in the regulation of IL-10 with the development of cancers that are in communication with microbiota, such as colon cancer, where the overexpression of miR-671-5p has been linked with poor prognosis [16, 17]. Additionally, in some types of cancers, the immunosuppressive role of IL-10 could facilitate tumor immune escape. However, these findings need further experiments.