Circulating microRNAs in Idiopathic Pulmonary Fibrosis

1. Introduction

Interstitial lung diseases (ILDs) are a complex set of heterogeneous chronic lung diseases that are difficult to diagnose and classify and affect the lung parenchyma due to inflammation and fibrosis. They can be divided into two groups: ILDs of known cause, such as those related to systemic diseases of connective tissue, environmental exposure, radiation, occupational exposure, or allergens, and those classified as Idiopathic Interstitial Pneumonia (IIP). The most common and with the worst prognosis of these is idiopathic pulmonary fibrosis (IPF), which is characterized by progressive remodeling of the lung parenchyma, excess deposition of the extracellular matrix, and irreversible scarring, which prevents an adequate gas exchange [1,2,3].

2. Idiopathic Pulmonary Fibrosis

IPF is a chronic, progressive, and lethal disease with a survivor mean of 3-5 years after diagnosis. The causes and mechanisms of the development of IPF are unknown. It has been proposed that it is characterized by an epithelial-dependent process that starts with micro-injuries over time in alveolar epithelial cells (AECs), with subsequent aberrant activation of these cells. The disruption of the alveolar epithelial barrier causes hyperplasia of pneumocytes type II (cuboidalization) and apoptosis of AECs. The damage of AECs produces many kinds of profibrotic mediators like transforming growth factor beta 1(TGF-β1), connective tissue growth factor (CTGF), platelet-derived growth factor (PDFG), etc., that promote profibrotic micro ambient in the lung of IPF patients. The above promotes an increase in migration, proliferation of lung fibroblasts, and differentiation in myofibroblast (activation). These cells produce excessive quantities of extracellular matrix (ECM) proteins like collagens type I and III, which finalize with the destruction of the pulmonary architecture (Figure 1). [4,5,6,7,8]

Risk factors associated with the development of IPF include a history of smoking, gastroesophageal reflux, chronic viral infections such as Epstein Barr and hepatitis C, a family history of interstitial lung diseases (rare variants in some genes such as MUC5B, TERT SFTPC, etc.), and aging. Symptoms begin insidiously with chronic exertional dyspnea, cough, inspiratory bibasilar crackles, and digital clubbing without symptoms that suggest a multisystem disease[9,10,11].

The natural history of IPF is heterogeneous; it can present chronic stable symptoms up to an acute respiratory exacerbation, with worsening dyspnea from days to weeks, which is associated with a poor prognosis, causing 40% of the deaths in patients with IPF. [12]

One of the presentations of IPF is rapidly progressive, in which patients present a decrease in forced vital capacity (FVC) and diffusion capacity of the lung for carbon monoxide (DLCO) of ≥10% and ≥15%, respectively, within the first 12 months after diagnosis, whose prognosis is severe. The diagnosis of this disease and its differentiation from other ILDs is challenging. It is essential to differentiate it accurately and promptly since inadequate treatment can increase the morbidity and mortality of patients. [10,13]

3. Serum/Plasma Biomarkers in IPF

Several proteins have been measured in the serum of patients with IPF to discover biomarkers to diagnose and prognostic for IPF. It is necessary to find a biomarker due to the difficulty in diagnosing IPF from other interstitial lung diseases like hypersensitivity pneumonitis, sarcoidosis, etc. In this context, some proteins evaluated as possible biomarkers of IPF include surfactant proteins A and D (SP-A and SP-D), Krebs von den lungen 6 ( KL6), C-C motif chemokines (CCL16, CCL18), matrix metalloproteinases (MMP7, and MMP28), renin and soluble (pro) renin receptor. [14,15,16,17]. The only promising molecule was MMP7, which has different levels between IPF and other ILDs. Another kind of molecule that has been measured in the serum of patients is micro RNAs, which are opening new ways to identify biomarkers for IPF (Figure 2).

4. miRNAs, Biogenesis, and Mechanism of Action

miRNAs are small RNAs of a length of 12-18 nucleotides (mature miRNA). This RNA regulates many RNA messengers (mRNA) post-transcriptionally in two ways: inhibition of mRNA translation or inducing its elimination. The miRNAs are non-coding RNAs found in the genome [18]. The miRNAs are transcribed by RNA polymerase II (RNA pol II), generating an immature miRNA termed pri-miRNA, which needs to be processed until it becomes a mature miRNA[19]. The mechanism described to produce a mature miRNA begins when miRNA is transcripted by RNA pol II. This pri-miRNA is processed by the RNAse type III Drosha and DGCR8 (DiGeorge syndrome critical region 8) complex, producing pre-miRNA of the 70 nucleotides in length [20,21,22]. Pre-miRNA is exported outside the nucleus using the nuclear pore complex RAN GTP/exportin 5[23,24]. In the cytoplasm, this pre-miRNA is taken by a complex formed by Dicer/ protein kinase R/ PACT that cut, generating the mature miRNA (Figure 3). Subsequently, mature miRNA is directed to its

RNAm target through the miRISC protein complex that consists of transactivating response RNA binding protein (TRBP) that, in turn, recruits Argonaute 2 (AGO2), followed by the coupling of GW182[25,26,27,28,29]. The miRISC, through AGO2, selects one of the two strands and then is directed to the RNAm target [30].

miRISC leads to the miRNA at the 3’untranslated region (3’UTR) of the RNAm, where the seed region is joined through Watson and Crick complementarity. This process has two results. 1) Depending on the kind of RNAm, the translation of the RNAm will be inhibited either at the level of preventing the formation of the complex of pre-initiation of translation (eIF4G, ribosomal subunit formation) or once the translation process has been started the miRISC complex plus miRNA joined to the 3’UTR region will promote decoupling of the translation machinery[26]. 2) The interaction of miRNA with target RNAm will produce the degradation of RNAm [25,31].

5. Circulation of Exosomes and Their Mechanism of Action

Exosomes can host different biomolecules, including nucleic acids (mRNA and miRNA) and various proteins, including enzymes, receptors, and transcription factors [32,33,34,35,36].

The biogenesis of exosomes begins through the endocytosis of biomolecules inside the cell through the invagination of the plasma membrane, forming the early-staging endosome (ESE)[37]. However, they can follow two routes; the first is to form “recycling endosomes,” and the second consists of subsequently undergoing a process of maturation to become late-classification endosomes (LSE) [38,39], which finally leads to the formation of multivesicular bodies (MVBs). Its formation consists of a second intussusception within the endosomal membrane, thus forming the intraluminal vesicles (ILVs)[40].

Therefore, MVBs are ILV-rich compartments that will eventually become exosomes upon released into the extracellular space. While there must be a fusion between the MVB and the plasma membrane to release the ILVs, there is also degradation by lysosome fusion or autophagosomes (Figure 4) [41,42].

The molecules internalized in exosomes do not appear random; instead, the load is characteristic of the conditions of origin[43]. We will focus on microRNAs because they are in post-transcriptional regulation[44]. The expression levels of these exosomal microRNAs have been reported in pathophysiologies or different tissues of origin[45].

The machinery by which microRNAs are internalized in exosomes and subsequently released depends on the machinery of the endosomal sorting complex required for transport (ESCRT), a protein complex involved in packaging biomolecules in the LVIs of MVBs [46].

MVBs can release exosomes through several systems, the main one being via fusion with the plasma membrane and subsequent release of the exosomes into the extracellular space. In addition, by exosomal binding to and endocytosis of target cell receptors (Figure 5). [47]

6. Upregulated Serum/Plasma miRNAs in Idiopathic Pulmonary Fibrosis

• miR-21

Many circulating miRNAs have been reported in IPF (serum/plasma); some are overexpressed, and others are downregulated. One of the more constant miRNAs with scientific evidence in IPF is miR-21, which has been found elevated in serum/plasma samples from IPF patients inclusive of proposed like predictor prognosis [48,49,50]. miR-21 The TGF-β1 signaling induces a positive loop because it increases the expression of miR-21; it has been reported that the SMADS (2,3,4) regulate the promoter activity of miR-21[51].

• miR-155

Levels of serum miR-155 in IPF are elevated compared with the control group [48,50]. miR-155 is related to the development of pulmonary fibrosis in several models in vitro and in vivo. It has been reported that miR-155 increases in mice bleomycin model and lung fibroblasts increase migration through Keratinocyte Growth Factor[52]. Besides in the silicosis model in mice, miR-155 has profibrotic effects by inhibition of mephrin α; this protein has antifibrotic effects because stimulation with mephrin α decreases expression of TGF-β1, and the receptors of TGF-β1 (TGFBRI and TGFBRII)[53]. The inhibition of miR-155 in a bleomycin model resulted in diminished profibrotic effects that downregulated the TGF-β1 and IL4 (interleukin 4) [54]. In the same way, as in other types of pulmonary fibrosis derived from systemic sclerosis, miR-155 is also elevated and is required for collagen synthesis during the fibrotic process[55]. It has been proposed that this mechanism is promoted by miR-155 inhibition of the protein forkhead box O3 (FOXO3a), resulting in the activation of the NLR family pyrin domain containing 3 (NLRP3)[56]. However, there is contrary evidence about the role of miR-155 in the development of pulmonary fibrosis, where it has been reported that miR-155 inhibits epithelial-mesenchymal transition (EMT) by targeting the glycogen synthase 3 beta (GSK-3β) and its interaction with liver X receptor (LXR) to exert antifibrotic effects [57].

• miR-590-3p

miR-590-3p is elevated in the plasma of patients with IPF, but there is no direct evidence in some models of pulmonary fibrosis. However, reports in other tissues suggest that the effect of this miRNA depends on the location[58]. For example, in heart fibroblasts, miR-590-3p regulates the proliferation, migration, and collagen synthesis by regulating the protein zinc finger E-box-binding homeobox 1(ZEB1) [59]. In contrast, miR-590-3p inhibits the protein mouse double minute 2 homolog (MDM2) in hepatocellular carcinoma, blocking EMT[60]. It is not clear the effect of miR-590-3p in the development of pulmonary fibrosis. Perhaps the effects depend on the location; this effect has been reported in other molecules in IPF, like matrix metalloproteinase 1 (MMP1), where the cellular location is important. MMP1 is over-expressed in epithelial cells of IPF but not in lung fibroblasts, where it is diminished[61]. It has SMAD7 as a target, an important protein in the signaling pathway of TGF-β-1 due to SMAD7 being an inhibitory protein of the SMAD2/3[62,63]. This regulation is crucial because TGF-β1 signaling can trigger several molecular events in IPF, like an increased expression of ECM proteins (collagen I type I, collagen III, etc.), induce fibroblast proliferation, EMT, etc. [64,65,66]. miR-21 has been consistently observed to promote lung fibrosis in fibroblasts and AECs. Another target of miR-21 is the protein phosphatase and tensin homolog (PTEN), which miR-21 induces EMT in a fibrosis model of ionizing radiation[67,68,69]. Resveratrol has been reported as an antifibrotic agent, and miR-21 could reverse the effects of resveratrol in a bleomycin model in rats, and in a contrary sense, treatment in rats with bleomycin diminished the expression of miR-21[70].

• miR-199a-5p and miR-200c

miR-199a-5p had the more consistent results because it was first reported elevated in a bleomycin model of lung fibrosis and tissues from IPF. Second, serum IPF levels of miR-199a-5p are elevated; interestingly, exosomes extracted from urine have the same result[71]. miR-199a-5p is elevated in the mouse bleomycin model, and the reported targets of miR-199a-5p are caveolin-1 and sestrin-2 (SESN2). Loss of caveolin expression has been associated with lung fibrogenesis, and levels of caveolin-1 are downregulated in the lung tissue of IPF patients[72,73,74,75,76]. SESN2 has been reported to have an anti-fibrotic effect in a model of stimuli with eupatilin; TGFβ1 induces the reduction in the production of the protein of SESN2 sestrin2[77]. In addition, miR-199a-5p has been related to regulating the mesenchymal stem cell senescence in IPF[78]. Another effect was reported in liver fibrosis, in which the inhibition of miR-199a-5p diminished fibrosis[79].

miR-199a-5p has targets like activating transcription factor 6 α (ATF6α) and inositol requiring enzyme 1 (IRE1); both have been reported to be sensors of endoplasmic reticulum stress (ERS) [80,81]. Levels of ATF6α and IRE1 are elevated in lung fibrosis (bleomycin and silica models) and in tissue sections of IPF, which is contradictory according to elevated levels of miR-199a-5p. However, in mice models of cardiac fibrosis, the absence of ATF6α in cardiac fibroblasts contributes to the augment of profibrotic fibroblast markers like collagen 1 and alpha-smooth muscle actin in mice model [82].

miR-200c is elevated in serum from IPF patients and is associated with interstitial lung abnormalities[71]. In contrast, miR-200c is reduced in the lung fibrosis of the bleomycin model, and epithelial cells after treatment with TGF-β1 and diminished of miR-200c was related to the development of EMT and loss of ability to alveolar epithelial cells type II to differentiate into alveolar epithelial cells type I [83,84,85]. The process correlates with another target of mir 200c, cadherin 11 (CDH11), which is elevated in lung fibrosis and is related to fibroblast migration, myofibroblast differentiation, and the EMT process during lung injury [86,87,88]. Another example in which reduced miR-200c expression is relevant is because it regulates fibronectin, a component of the MEC that accumulates in the lung fibrotic process[89,90]. miR-200c seems to participate in regulating EMT through ZEB1[91]. These examples suggest that miR-200c levels are downregulated in IPF.

7. Downregulated Serum/Plasma miRNAs in Idiopathic Pulmonary Fibrosis

Let-7a

and Let-7d.

Let-7a and Let-7d are downregulated in serum samples from patients with IPF and have the same targets related to the development of lung fibrosis via TGF-β1 signaling pathway like protein high mobility group AT-hook 2 (HMGA2). HMGA2 is a chromatin protein that regulates gene transcription, participating as a co-factor for other transcription factors. Have been reported that let-7a and let-7d could suppress EMT and fibroblast proliferation by blocking HMGA2[71,92,93,94]. In the same signaling pathway, the targets of let-7a and let-7d are the receptors of TGF-β1 (TGFBR1 and TGFBR3) [95,96,97]. Let-7a and let-7d play important roles in developing pulmonary fibrosis by regulating several components of the TGF-β1 signaling pathway.

Then decreased expression levels of let-7a and let-7d suggest that they participate in promoting pulmonary fibrosis in IPF because they have other profibrotic targets like insulin-like growth factor receptor (IGF1R), platelet-derived growth factor B (PDGFB), both receptors are increased in lung fibrosis. Another profibrotic target of let-7a is the activin A receptor type 1B (ACVR1B), which has been implicated in the pathogenesis of cardiac fibrosis through activation by Activin A. Human lung fibroblasts express the receptors of activin A (ACVR1B included) and have a profibrotic effect by promoting collagen contraction, which is a crucial event inside of development of lung fibrosis [98,99,100,101,102].

• miR-16

Serum miR-16 is downregulated in IPF, and this miRNA has antifibrotic activity[103]. miR-16 inhibits the activity of TGF-β1 and collagen I expression in dermal fibroblast; this inhibition is attributed to the target of miR-16 being SMAD3[104]. In hepatic stellate cells, miR-16 suppresses proliferation and fibrogenesis provoked by TGF- β1 regulating lysyl oxidase homolog 1 (LOX L1)[105]. Other evidence about the antifibrotic effects of miR-16 is found in systemic sclerosis, where it suppresses myofibroblast activation by inhibiting neurogenic locus homolog protein 2(NOTCH2) [106]. HMGA1 and HMGA2 also are targets of miR-16, fibroblast growth factor receptor 1 (FGFR-1), IGF1R [107,108,109]

• miR-25-3p and miR-142-5p

miR-25-3p is downregulated in pulmonary fibrosis, but there is no data about its role in lung fibrosis; it has been reported that miR-25-3p expression is increased in cardiac fibrosis, and its expression is related to the augmentation of alpha-smooth muscle actin[110,111]. In a contrary sense, in hepatic stellate cells, the overexpression of miR25-3p has a positive effect because of the Notch 1 signaling pathway and profibrotic results in the induction of collagen expression through TGF-β1 [112]. Additionally, there is a report that the inhibition of miR-25-3p indirectly caused the elevation of TGF-β1. These findings reinforce the relationship between miR-25 and the TGF-β1 signaling pathway [113]. miR-142-5p is downregulated in serum from IPF, but in macrophages from broncho-alveolar lavage fluid (BALF) of patients with IPF, miR-142-5p seems to be elevated, boosting profibrotic actions in vitro like M2 polarization, production of profibrotic cytokines like C-C motif chemokines (CCL18, CCL17, CCL13), TGF-β1, IL4 that in turn influence neighboring fibroblast[50,114].In the same sense, a blank reported of miR-142-5p is the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor, which is considered antifibrotic because its actions negatively regulate the TGF-β1 signaling pathway[115,116,117]

• miR-101-3p

miR-101-3p is downregulated in serum and lung tissue sections of patients with IPF. This is important because miR-101-3p has been reported to regulate the WNT5a signaling pathway, which reduces proliferation in lung fibroblasts by regulating the receptor frizzled-4,6 (FZD4/6). WNT5a is elevated in IPF, and its signaling pathway has many profibrotic effects, such as inducing fibroblast proliferation, increasing resistance to apoptosis, and increasing fibronectin expression. miR-101-3p also has like blank to the TGFBRI receptor of the TGF-β1 signaling pathway, and it seems that miR101-3p regulates both signaling pathways WNT5a and TGFβ1 in a coordinated manner [50,118,119,120,121]. Another target of miR-101 with profibrotic effects is endothelin-1; this protein is elevated in the serum of patients with IPF and has many profibrotic effects like inducing production of collagen I and III, overproduction of CTGF, fibronectin, α-smooth-muscle actin[122,123,124,125]

8. Common Targets in TGF beta-1 Signaling Pathways

TGF-β1 is a profibrotic molecule that triggers various molecular events in the development of pulmonary fibrosis, and such events affect both alveolar epithelial cells and lung fibroblasts [126]. In the case of epithelial cells, TGF-β1 induces EMT, and activation of the AECs results in the production of many profibrotic molecules like plasminogen activator inhibitor 1 (PAI-1), CTGF, PDGF, etc.[66,101,127,128]. Lung pulmonary fibroblasts affected by the TGF-β1 signaling pathway are activated, which causes an increase in the expression levels of collagen type I and III, induced proliferation, and fibroblast differentiation to myofibroblast (augmentation of α-smooth muscle actin) that is more aggressive in collagen deposition, and this stimulus gives them resistance to apoptosis[129,130,131,132,133,134,135,136].

miRNAs let-7a, let-7d, miR-16, and miR-25-3p are downregulated in serum samples from patients with IPF and PCR-validated, according to Tarbase 9.0 is a database to deliver experimentally supported miRNA targets on RNAm from AGO-CLIP-seq protocols that included for example HITS-CLIP, PAR-CLIP. Interestingly, this miRNA shares targets of the TGF-β1 signaling pathway [137]. For example, let-7a, let-7d, miR-16, and miR-25-3p aim at the same target: histone acetyltransferase CREBBP. This protein has been related to promoting an increase in the expression of collagen VI, which is augmented in lung fibrosis and participates in EMT, an important process in developing IPF.[138,139,140]. Let-7d and miR-16 share SMAD3 as a target [70]. SMAD 2 is regulated by let 7d, miR25 and miR 16[141,142]. Let-7d, Let-7a, and miR-25-3p regulate the receptor TGFBR1[97]. Let 7a and Let 7d have like targets of TGFBR2 and AVR1B receptor are regulated by Let 7a, Let 7d and miR16 (Figure 6)[143,144]

9. Discussion and Conclusions

Serum/plasma biomarkers in IPF have been investigated several decades ago[145]. The importance of finding noninvasive biomarkers is due to the characteristics shared between IPF and other ILDs (like hypersensitivity pneumonitis), and therefore, the differential diagnosis is complicated for clinicians[9,146]. miRNAs have emerged as another molecule that could be evaluated in the serum/plasma of IPF patients a few years after the first report about miRNAs and vesicles like exosomes [35,48]. Due to the miRNAs, they could move through the bloodstream protected by exosomes and with protein Argonaute 2 complexes and posteriorly detected by their isolation through the total RNA extraction and its subsequent real-time PCR evaluation. In the field of ILDs, miRNAs in the serum /plasma of IPF patients have only been evaluated compared with the control group[147,148].

Generally, let-7a and let-7d are downregulated, and miR-21 is elevated in IPF. The results have been reported consistently in serum and lung fibroblasts compared to other miRNAs evaluated in serum/plasma. But today, there is no report about serum miRNAs levels in other ILDs like hypersensitivity pneumonitis to find some miRNA differentially expressed with IPF[48,49,50,51,71,92].

The search for miRNAs in serum/plasma is to correlate the expression levels with the clinical characteristics of the patients to find a relationship with the pulmonary state of IPF and to identify a biomarker that could serve as diagnostic or prognostic [149,150]. Additional information obtained indirectly from the serum/plasma miRNAs profile is to link signaling pathways in which they are involved through the in-silico analysis of reported target mRNAs[151]. In addition to the above, the serum miRNA analysis provides new signaling pathways that could participate in the pathogenesis of IPF by using software that predicts new targets of this type of RNA[152].

The contradictory results regarding miRNAs levels in serum/plasma compared with levels in lung tissue sections and fibroblasts from patients with IPF could be explained by the effect of regulation of long non-coding RNAs that have the function of regulating (sequester) the miRNAs[153]. The above responses partially explain why a miRNA is elevated in serum/plasma, but its target is unaffected. Another aspect to consider in regulating the function of miRNAs is the RNA edition mechanism carried out by ADAR proteins. This process could affect 1) the rate of the miRNA processing and redirection of miRNA to another target mRNA[154].