In this study, we investigated plasma miR-21, miR-590, miR-192, and miR-215 levels in IPF patients. The plasma miR-21 and miR-590 levels in IPF group were significantly higher than those of the control group, but there was no significant difference in miR-192 and miR-215 levels. Moreover, miR-21 and miR-590 were associated with age and DLCO, while miR-192 and miR-215 were associated with age, GAP score, and honeycombing. These findings suggested that miR-21 and miR-590 could take a role in the development of IPF, additionally, miR-192 and miR-215 also could be related to the progression of the IPF.
MiRNA molecules have been reported to be involved in lung fibrosis. Formerly, the expression level of miR-21 was found to increase both in the lung tissue of bleomycin-treated mice and in the lungs of IPF patients [5]. Then, the serum miR-21 was suggested as a representative of miR-21 expression in the lung tissue of IPF patients [15], and additionally, increased serum miR-21 levels were reported in two different IPF studies [7, 16]. In our study, the plasma miR-21 levels were significantly higher in IPF than in the control. Together with former studies, our results provide further evidence that miR-21 levels are elevated in plasma of the patients with IPF.
To the best of our knowledge, there is no data about the plasma levels of miR-590, miR-192, and miR-215 in IPF. Each one of these miRNAs has been reported to be involved in various fibrotic diseases but only miR-192 was associated with lung fibrosis [10, 17, 18]. MiR-192 expression was found to be increased in the bleomycin-induced fibrotic lungs of mice and in the sputum of IPF patients [11, 12]. Additionally, miR-192 has been shown to be a modulator of airway remodeling in patients with asthma [13]. In this study, the plasma miR-590 levels were significantly higher in IPF than in the control, while the expression levels of both miR-192 and miR-215 were similar.
Besides the presence of IPF, our study provided evidence that plasma miRNAs could display expressional alterations with the severity of IPF. The mean plasma level of miR-590 was high in IPF group, but it was decreased as the DLCO values decreased. This data suggests that plasma miR-590 expression increases with IPF development, but along with the disease progression it reduces. Thus, the suppression of miR-590 expression could be related to a negative feedback mechanism associated with the continuing fibrotic processes. Moreover, plasma level of miR-192 did not change by the presence of IPF, but it was decreased as the radiological severity or the GAP score increased. So, miR-192 could be a promising plasma biomarker for the prognosis of IPF.
In this study, miR-21 was positively correlated with miR-590 in both control and IPF groups. Previously, they were observed to display an inverse expression pattern in ulcerative colitis and there was no correlation between them [19]. Furthermore, our results showed that both miR-21 and miR-590 positively correlated with age in IPF group. The circulating levels of miR-21 were previously reported to increase with age and the miR-590 levels were also found elevated in older subjects [20]. Supporting these data, miR-21 was reported as DNA damage-related circulatory biomarker for aging [21]. Interestingly, we observed that miR-192 and miR-215 were negatively correlated with age in IPF group. Although circulating miR-192 levels were suggested to decrease with age, miR-21 and miR-192 were found elevated in circulating vesicles of aged mice [22]. Since miRNA molecules in the circulation are distributed in several compartments as protein-bound or in exosomes, currently it’s unknown whether these different compartments are particularly associated with the specific diseases [23]. Importantly, the effect of age on miRNA levels seems to be depending on the severity of the current disease and the specific compartment of miRNAs in which they are distributed.
The interaction of miRNAs with fibrotic processes could be associated with the fibrotic signaling pathways. TGFb molecule, closely related to miR-21, is an important factor in pulmonary fibrosis. In renal fibrosis, TGFb induces fibrosis with Smad3 directed upregulation of miR-21, while miR-21 modulates TGFb signaling by reducing the inhibitory Smad7 molecule. Lung injury stimulates miR-21 and TGFb expression and also inhibits Smad7 expression [24]. During bronchial airway remodeling, TGFb induces miR-21 expression but then miR-21 inhibits Smad7 and further stimulates TGFb signaling [25]. Collectively, miR-21 seems to be a positive feedback regulator of TGFb signaling in fibrosis development.
Together with miR-21, miR-590 has also been implicated in the fibrotic TGFb signaling. Although TGFb reduces miR-590 expression, miR-590 directly inhibits both TGFb and TGFb receptors to suppress its signaling pathway, but miR-590 also inhibits Smad3 and Smad7 expressions [26–28]. Therefore, it has been suggested that miR-590 and TGFb molecules act as negative regulators of each other. However, these data are in contrast with our observation of a higher plasma level of miR-590 in IPF patients than in healthy subjects. A possible explanation for this discrepancy might be associated with the inflammatory response. Pro-inflammatory cytokines regulate miR-590 expression by JAK/STAT pathway [29]. During inflammation processes, STAT3 activates STAT5, and STAT5 is reported to increase miR-590 expression, whereas STAT3 is identified as a target of miR-590. In addition, STAT3/5 proteins reported to be involved in the regulation of EMT with TGFb signaling in pulmonary fibrosis [30]. These data suggest that miR-590 might have a dual role in both inflammation and fibrosis during IPF progression as a negative feedback regulator.
Interestingly, TGFb-induced miR-192 functions are much more heterogeneous, miR-192 can inhibit or stimulate fibrosis depending on the cell type. TGFb signaling with Smad3 promotes miR-192 expression, whereas Smad7 reduces miR-192 expression. Previously, miR-192 expression was found reduced in the cigarette smoke-exposed lung tissue. However, during bleomycin-induced pulmonary fibrosis, miR-192 expression was increased at first but reduced later [11]. Also, high levels of exosomal miR-192 were found in the sputum samples of IPF patients [12]. Altogether these data support that the effect of TGFb on fibrosis via miR-192 differs according to the target cell type and miR-192 may play a regulatory role in IPF disease development and progression.
There are limitations in this study. The control group was small in number and the correlation of target miRNAs with age in control group needs to be analyzed with further studies. In addition, it’s not possible to know whether miRNAs in the plasma of IPF patients are originated from lung tissues or extra-pulmonary tissues, or circulating cells.
In conclusion, the plasma miR-590 and miR-21 levels were found significantly higher in the IPF group than in the control group. Moreover, the miR-21 and miR-590 levels were found to be related to age and DLCO, while miR-192 and miR-215 levels were associated with age, GAP score, and honeycombing. Therefore, these miRNAs may take a regulatory role in the development and progression of IPF and may be utilized as a diagnostic or prognostic tool or potentially, as a new therapeutic target. Nevertheless, further research is needed to better understand the roles of plasma miRNAs in IPF.