The expression of 15 miRNAs previously identified as differentially regulated between myometrial PCs (MPCs) and leiomyoma PCs (PCs) [5] was compared to that observed in Amniotic Fluid PCs (AFPCs) to evaluate if the dysregulation of the 15miRNAs occurs: i) as the result of an early commitment of undifferentiated cells (Fig. 1A) or ii) as an exacerbation of the physiological differentiation from AFPCs to MPCs (Fig. 1B). The regulation of the expression level of the miRNAs across the cell type analysed does not allow giving unique suggestions.
Firstly, cells isolated from healthy and fibrotic uterine tissues as well as from amniotic fluids were characterized according to the minimal criteria established by the International Society for Cellular Therapy (ISCT) for the identification of human mesenchymal stem cells7and were able to satisfy them.
After characterization, the first issue to address was the validation of the choice of AFPCs as the most undifferentiated cells to be used as a surrogate of the embryonic ones [9, 19]. Consistent with the fact that AFPCs are the most undifferentiated cells among the three cell types analyzed, they cluster separately from MPCs and LPCs that are instead closer to each other.
The heat map and the histogram show that the expression of the majority of 15 miRNAs is very different between AFPCs and LPCs/MPCs (mean of absolute FC 2.6 ± 2.8). Clustering analysis followed by DIANA mir-Path investigation allowed identifying four KEGG pathways related to the miRNA-clusters defined based on expression: Adherens junction, ECM-receptor interaction, TGFβ signaling and cell cycle.
To address the question of which hypothesis was more consistent (A, early commitment; B, exacerbation), the trend of expression across the cell types of each identified pathway was evaluated. Only adherens junction and TGFβ signaling pathways show a step-wise manner along the differentiation process.
To investigate what is the most probable hypothesis explaining the origin of LPCs, (A, early commitment; B, exacerbation), we evaluated in details the pattern of miRNA expression across the three cell-types. Only the 4 miRNA linked to regulation of adherens junction and one liked to TGFβ signaling pathways (miR-122-5p) show a progressive decreased or increased expression (respectively) from AFPCs to LPCs, ie along the hypothetical differentiation process.
Interestingly, LPCs expressed the lowest values of miRNAs related to adherens junction pathway and, most-likely as consequence, also the highest level of targeted proteins. As expected, the expression of adhesion molecules is higher in connective tissues where fibroblasts/myofibroblasts develop strong bounds to ECM than in AFPCs [20]. While the increase in the expression of proteins related to adherens junction from AFPCs to MPCs has to be considered physiological, its further weighting towards LPCs may be connected to the leiomyoma onset [21, 22].
Notably MED12, implicated as an oncogene in about 70% of uterine leiomyoma [23, 24], is a target of hsa miR-10a-5p, whose expression is gradually reduced from AFPCS up to LPCs, possibly explaining MED12 upregulation in leiomyoma.
While the expression of the three miRNAs converging in TGFβ signalling pathway (has-miR-122-5p; has-miR-576-3p; has-miR-595) is weakly increased in MPCs compared to AFPCs, they are strongly upregulated in LPCs. The TGFβ pathway has been reported as deeply dysregulated in leiomyoma [25–27]; the genes targeted by these miRNAs belong to a cascade that involves SMAD 2 and SMAD4 and leads to apoptosis and G1 cell cycle arrest; the high expression of the selected miRNAs in LPCs may intervene by suppressing these proteins. This is in line with previous researches [27] reporting that leiomyomas are refractory to the antiproliferative effects of TGFβ1 and TGFβ3 observed in normal myometrium.
For the other two pathways, the expression of miRNAs across the cell type does not show a linear trend that could reinforce the hypothesis of exacerbate differentiation from MPCs to LPCs. Previous studies about miRNA profiling from tissues samples of fibroid and myometrium identified a list of involved pathways that encloses the four here considered [28].
Interestingly, even if our pathways are enclosed in the list, the identified miRNAs are different.
It is not surprising that more miRNAs target the same genes but that the same pathways have been found differentially regulated in progenitor as well as in differentiated cells confirms the involvement of the selected pathways in the onset of leiomyoma. Indirectly, it also strengthens the hypothesis that the modulation of the expression of miRNAs during differentiation is a key mechanism in the pathogenesis of leiomyoma.