The most indicative result of the study is the variation of miR-16 and let-7d relative expression compared to healthy controls. This result was expected for let-7d, that has a huge literature support for [9, 26], but it is completely new for miR-16, considering no previous studies have indicated a relationship between interstitial lung diseases and down-regulation of miR-16 expression. Pandit et al. [27] assigned to miR-16 an antifibrotic function, even if there are only scientific evidences on hepatic fibrosis. In this context, miR-16 is thought to be an Ito cell quiescence maintaining factor [19]. Actually, miR-16 could have the same role on AEC-II and fibroblasts that, in response to damage and chemo-cytokine stimulation, can behave like Ito cells. A consideration to make is that the exosome content is not strictly related to the component concentration in the body fluids or in the intracellular environment but is a sort of “packed information” that the producing cell wants to provide to the receiving cell. This can be a possible explanation for the non-significant decrease of miR-26a, that theoretically would have had the same results of let-7d. Anyhow, the high relationship found between miR-26a and let-7d expression seems to confirm the dysregulation in this cellular pathway [9].
In our study we detected a down-regulation of the relative expression of all the 5 miRNAs analysed. If the reduction in miR-16, miR-26a and let-7d can be related to the malfunction of regulation mechanisms potentially leading to the onset of IPF, the same explanation is not applicable for miR-21 and miR-210. We could just make hypotheses to explain these results.
Obviously, although miRNAs are employed for exosomal cell signaling, many miRNAs remain in the cell. Those passing into exosomes do so via a loading process that has not been completely understood. A first intuitive and simplistic explanation could be that those miRNAs, although augmented in the cell, are partially excluded from the composition of exosomes. It is worth noting that the composition of the exosomal content not necessarily reflects the concentration of the same molecules in the cell or in body fluids.
The second hypothesis is that the down-regulation of profibrotic miRNAs could be the result of a compensation mechanism put in place by healthy cells in order to contain the pathology. To confirm this hypothesis, we should have obtained an up-regulation of anti-fibrotic miRNA or at least a similar expression level to the housekeeping miRNA of healthy control. This did not occur, so that the current hypothesis is just a suggestion for further studies.
A third hypothesis could be that, actually, miR-21 and miR-210 are not the main key players in the trigger and/or the maintenance of the fibrotic process. Therefore, the expression of such profibrotic miRNAs might be decreased to avoid the same cell or other physiologic players to detect the profibrotic stimulus and react, re-establishing an anti-fibrotic environment.
The last result of our study to focus on is the significant clustering of miR-16, miR-21 and miR-26a. At this regard we have to specify thatexosomal miRNA is only a part of the whole miRNA present in serum. Since the nude RNAs in the blood are degraded in a short time being readily targeted by the exonucleases that are widely present, they must be protected from degradation. In addition to the mechanism of exosomes, miRNAs are packaged and transported in plasma also through detachment of micro-vesicles (MV) and apoptotic bodies, as well as by proteins that bind RNA and HDL. Therefore, in order to confirm and study in depth this outcome, a comparison of the same samples both for the exosomal miRNA expression and for the serum one it would be needed. On the other hand, there were no relationships between the expression of the miRNAs being studied and the clinical characteristics of the enrolled patients. Further studies are thus mandatory to better identify, whether a dependency with the severity of the disease actually exist, specific alteration in miRNA’s clusters that may help to explain in detail the evolution of the pathology. Moreover, a limit of this study was the use of only miRNA-222 as endogenous control. Probably, this choice could influence the expression of other miRNAs tested. It is true that miR-222 shows a differential expression in IPF, as reported by Sameer R Oak et al. [28], but in this study miRNAs were isolated from surgical lung biopsies of IPF patients instead of from sputum [22], plasma [29] and serum as we did in this study. Overall, these studies, including ours, indicate that the knowledge of miRNA expression in IPF is still in its infancy and caution should be posed by comparing studies that investigate different samples for studying these biomarkers. Nevertheless, this is a preliminary study pilot study that will be followed by a larger one and we are planning to evaluate this aspect in a future study.