Background
Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease for which no treatment is capable of providing a complete cure [
1‐
3]. The median survival for IPF patients from the time of diagnosis is approximately 3 years [
3]. Recently, new therapeutic targets for IPF have been identified, and some of the proposed therapies are expected to slow its progression [
2]. IPF patients differ in terms of the disease progression rate and prognosis, complicating the prediction of survival. The identification of prognostic predictors for IPF is important for determining who requires the most intensive therapies.
MicroRNAs are 22-nucleotide-long non-coding RNAs that function in the translational repression or degradation of target mRNA [
4]. MicroRNAs have been shown to affect physiological and pathological conditions, including lung disease [
5]. A recent investigation of fibrotic lung diseases showed that the expression levels of several microRNAs were significantly altered in fibrotic lungs, suggesting that microRNAs contribute to the development and progression of fibrotic lung diseases [
5]. Therefore, microRNAs have received considerable attention as potential therapeutic targets in IPF, as well.
Exosomes are one of the major components of extracellular vesicles (EVs) [
6]. Recently, EVs, including exosomes, have been considered as novel tools for intercellular communication because EVs contain various proteins and nucleic acids including microRNAs [
7]. MicroRNAs in EVs can be transferred to target cells to regulate gene expression and cell function [
8‐
10]. EVs and enveloped microRNAs have been shown to function in physiological and pathological conditions [
11‐
15]. EVs and enveloped microRNAs within biological fluids (e.g., circulating blood) have also attracted attention as novel biomarkers of diseases such as cancer because the components and secretion dynamics of EVs vary according to their cellular origin and environment [
16,
17].
In this study, we explored the possibility that microRNAs of serum EVs changed during lung fibrosis and could serve as prognostic biomarkers of IPF. We examined the levels of serum EV microRNAs in a mouse model of lung fibrosis via quantitative PCR array, which revealed that miR-21-5p was significantly increased in serum EVs of the mouse model. Accordingly, we evaluated the levels of miR-21-5p in serum EVs (serum EV miR-21-5p) after adjusting for difference in the quantity of serum EVs among IPF patients.
Methods
Reagents
Total Exosome Isolation reagent (Thermo Fisher Scientific, Waltham, MA, USA) was used for EV purification from serum. The miRNeasy Mini Kit and Syn-cel-miR-39-3p miScript miRNA Mimic for serum EV RNA purification was purchased from QIAGEN (Hilden, Germany). The following antibodies were used for ExoScreen detection of EVs; biotin-conjugated anti-mouse CD9 (clone MZ3, Biolegend, San Diego, CA, USA), anti-mouse CD9 (clone MZ3, Biolegend), and anti-human CD9 (clone 12A12, Shionogi & Co., LTD, Osaka, Japan). ChromaLink™ Biotin Labelling Kit (Solulink, Inc., San Diego, CA, USA) was used for biotinylation of anti-human CD9 antibody. AlphaLISA reagents (PerkinElmer, Waltham, MA, USA) for ExoScreen included AlphaScreen Streptavidin Donor Beads, unconjugated AlphaLISA Acceptor Beads and AlphaLISA Universal Buffer. Conjugation of AlphaLISA Acceptor Beads to anti-human or -mouse CD9 antibodies was performed according to the manufacturer’s protocol.
Cell cultures
A mouse lung cancer cell line (Lewis lung carcinoma (LLC)) and a mouse mesenchymal cell line (KUM-10) were obtained from RIKEN Cell Bank, Ibaraki, Japan. LLC and KUM-10 were cultured in DMEM with 10 % heat-inactivated fetal bovine serum (FBS) and an antibiotic-antimycotic solution (Thermo Fisher Scientific) at 37 °C in 5 % CO2.
Preparation of conditioned media and EVs in media
The cells were washed with phosphate-buffered saline (PBS), and the culture medium was replaced with advanced Dulbecco’s Modified Eagle Medium for LLC and KUM-10 cells. After incubation for 48 h, the conditioned media were collected and centrifuged at 2000 g for 10 min at 4 °C. To thoroughly remove cellular debris, the supernatant was filtered through a 0.22 μm filter (EMD Millipore, Billerica, MA, USA). To prepare EVs, the conditioned media were ultracentrifuged at 110,000 g for 70 min at 4 °C. The pellets were washed with 11 mL of PBS, ultracentrifuged at 110,000 g for 70 min at 4 °C and resuspended in PBS. The protein content of the putative EV fraction was measured using a BCA protein assay (Thermo Fisher Scientific).
ExoScreen assay
The detailed principle and analytical methods were presented in a previous report [
18]. Briefly, a 96-well half-area plate was filled with 5 μL of sample, 5 nM biotinylated antibodies and 50 μg/mL AlphaLISA acceptor bead-conjugated antibodies against mouse or human CD9 in the universal buffer. The volume of each reagent was 10 μL. The plate was then incubated for 3 h at room temperature. Without a washing step, 25 μL of 80 μg/mL AlphaScreen streptavidin-coated donor beads were added. The reaction mixture was incubated in the dark for another 30 min at room temperature, and the plate was then read on a PHERAstar FS microplate reader (BMG LABTECH, Ortenberg, Germany) using the AlphaLISA mode (excitation wavelength of 680 nm and emission detection set to 615 nm). Background signals obtained from PBS were subtracted from the measured signals.
The animal model of pulmonary fibrosis
All animal experiments were approved by the Tohoku University Animal Experiment Ethics Committee and performed in accordance with the Regulations for Animal Experiments and Related Activities at Tohoku University.
Seven- to 8-week-old male C57BL/6 mice were used in our experiments. C57BL/6 mice were purchased from CLEA Japan (Yokohama, Japan). All mice were housed in a specific pathogen-free facility and maintained under constant temperature (24 °C), humidity (40 %), and light cycle (8:00 A.M. to 8:00 P.M.), with food and water provided ad libitum. To induce pulmonary fibrosis, mice were treated intratracheally with bleomycin hydrochloride (Nippon Kayaku, Tokyo, Japan) on day 0 as described in our previous study [
19]. Briefly, mice were anesthetized with ketamine via intraperitoneal injection and were then injected with 0.04 mg of bleomycin hydrochloride in 100 μl of saline through a 27G needle inserted between the cartilaginous rings of the trachea. Circulating blood was harvested 7, 14 or 28 days after instillation. Collected blood was incubated at room temperature for 1 h and centrifuged for 15 min at 1500 g at 4 °C. The serum was transferred to a new tube and centrifuged again for 30 min at 2500 g at 4 °C to remove cells and debris. The clarified serum was transferred for further examination.
Subjects and specimens
This study was approved by the ethics committees at Tohoku University School of Medicine, Japanese Red Cross Ishinomaki Hospital and Hikarigaoka Spellman Hospital. All subjects gave written informed consent. This study is registered with UMIN-CTR, number UMIN000017403.
Human peripheral blood was obtained from patients or healthy volunteers at Japanese Red Cross Ishinomaki Hospital (Ishinomaki, Japan) or at Hikarigaoka Spellman Hospital (Sendai, Japan). Serum was obtained by centrifuging these specimens, aliquoted, and stored at −80 °C until used in analyses of serum EV. Forty-one patients with idiopathic pulmonary fibrosis (IPF) and 21 healthy controls were included (Table
3). Diagnoses were made according to the American Thoracic Society (ATS)/European Respiratory Society (ERS) statement based on clinical evaluation, high-resolution computed tomography, histology, and laboratory findings [
20]. Emphysematous lesions were detected by CT and evaluated with Goddard LAA score [
21]. The patients whose Goddard LAA score is ≥1 were considered as having emphysema. The expert clinicians who analysed the information were blinded to the diagnoses associated with the experimental laboratory tests. Usual interstitial pneumonia was confirmed by surgical biopsy in 15 of the IPF patients (Table
3). The patients were recruited at the time of diagnosis. The collection of blood samples and pulmonary function tests were performed at entry. IPF patients were followed for 30 months except in cases of death or failure to visit the hospital. Survival status was obtained from visits to the hospitals and telephone interviews. During the follow-up periods, the patients received pulmonary function evaluations 6 months after enrolment.
Preparation of EVs from mouse or human serum samples
Mouse or human serum samples were centrifuged at 10,000 g for 10 min to remove cells and debris. Subsequently, we extracted EVs from serum using a commercial extracting reagent (Total Exosome Isolation from serum, Thermo Fisher Scientific) as previously described [
22]. Briefly, we mixed 250 μL of centrifuged serum with 50 μL of an extracting reagent. The samples were incubated at 4 °C for 30 min and then centrifuged at room temperature at 10,000 g for 10 min. The supernatant was discarded and the EV pellet was resuspended in 100 μL of PBS. The EV suspension was used for further examination.
Extraction of serum EV RNA and synthesis of cDNA
Total RNA was extracted from serum EV using a miRNeasy Mini Kit (QIAGEN, Hilden, Germany). Synthetic C. elegans miR-39 (QIAGEN) was added to samples of the serum EV suspension to control for variations during the preparation of total RNA. The miScript II RT Kit was used for reverse transcription of microRNAs into cDNA.
MicroRNA PCR array in mouse serum EVs
The Mouse Serum miScript miRNA PCR Array (QIAGEN, MIMM-106Z) was used according to the manufacturer’s instructions for the comprehensive analyses of the expression levels of the microRNAs in the serum EV in a mouse model of pulmonary fibrosis. Briefly, template cDNA that had been synthesized from the mouse serum EV RNA was added to each well of the miScript miRNA PCR array plate. The plate was placed on the real-time cycler (StepOne Plus, Thermo Fisher Scientific) and run. The data were analysed using the web-based miScript miRNA PCR Array data analysis tool (QIAGEN). MicroRNAs were considered not detectable when Ct > 35. We also excluded the microRNAs for which the Ct > 30 in either group for further analyses on the basis that this indicated that the expression level was relatively low, which could cause greater variations in the fold-change results. The delta Ct value (target microRNA Ct–cel-miR-39 Ct) in each sample was calculated. The average of the delta Ct values in each sample group was then calculated and used to analyse the fold-changes of serum EV microRNAs compared with non-treated controls. The expression levels of microRNAs were normalized by dividing them by the amount of EVs in each serum EV sample, as determined by the ExoScreen assay. To control the false discovery rate for multiple comparisons, the Benjamini-Hochberg procedure was used [
23]. The
q-value for the fold-change for selecting the candidate microRNAs was less than 0.05.
Quantification of microRNAs in serum EVs
The microRNAs were quantified by real-time PCR using the miScript Primer Assay (QIAGEN). The real-time RT-PCR detection of the C. elegans miR-39 was also performed for the normalization of the real-time RT-PCR results of the endogenous microRNAs in the sample. This procedure corrects for variations during the RNA preparation, cDNA synthesis, and real-time PCR. To determine the copy numbers of human miR-21-5p in the serum samples, standard curves were prepared using serial dilutions of synthetic human miR-21-5p (Bioneer, Daejeon, Korea). The relative amount of the spiked cel-miR-39 in each sample was calculated using the serially diluted standard samples. This value was used for normalization between samples. The formula for this calculation is as follows: (The normalized amount of miR-21-5p or miR-155-5p in sample X) = (the pre-normalized amount of the microRNA in sample X) × (the amount of cel-miR-39 in the reference sample/the amount of cel-miR-39 in sample X). The reference sample was one of the non-treated control samples for mouse study or one of the IPF patient samples (the sample of IPF patient #1) for human study, respectively. The expression levels of the microRNAs were normalized by dividing them by the amount of EVs in each serum EV sample, as determined by the ExoScreen.
Statistical analysis
The statistical analysis was performed using JMP Pro 11.0 (SAS Institute Inc., Cary, NC). In the animal experiments, the data are presented as the means ± SEM unless otherwise indicated. The Kruskal-Wallis test was used for multiple comparisons, and
p < 0.05 was taken to represent statistical significance. We used the Benjamini-Hochberg procedure [
23] to control the false discovery rate for multiple comparisons for the microRNA PCR array analyses. The
q-value for the fold-change for selecting the candidate microRNAs was less than 0.05. In the clinical setting, the data are presented as the medians (IQR). The differences in the distribution of the categorical data between two groups were analysed using the Fisher exact test. The differences in continuous data between two groups were analysed using the Mann-Whitney
U test. Correlations between miR-21-5p and other clinical variables were calculated using the Spearman rank correlation. In the correlation analysis of the baseline characteristics, the Cox proportional hazards model was used to determine the effect of various factors on mortality in IPF patients. These results are expressed as hazard ratios for death among those who had a factor of interest compared with those who did not have the factor. We calculated the median values for the normalized or non-normalized serum EV miR-21-5p levels in the 41 IPF patients. These values were determined to be 2.1 copies/SI or 1.25 × 10
7 copies/mL, respectively. We divided the 41 IPF patients into the following two groups: those above and those below the median value. We also divided the IPF patients into the top-quartile versus the remaining subjects for the following analysis. Survival was evaluated using the Kaplan-Meier approach, and the differences in survival between two groups were compared using log-rank tests. A value of
P < 0.05 was considered to indicate statistical significance.
Discussion
This study first demonstrated that the expression profile of the microRNAs within the EVs in circulating blood was significantly changed in a mouse bleomycin-induced lung fibrosis model. Among the microRNAs that showed an altered expression during lung fibrosis, serum EV miR-21-5p expression was significantly upregulated in both the acute phase and the later chronic/fibrotic phase. In human subjects, the levels of serum EV miR-21-5p after adjustment for the quantity of EVs were significantly increased in the IPF patients. Moreover, the baseline levels of the serum EV miR-21-5p were significantly associated with the disease progression (the decline in the percent-predicted VC) and mortality. This is the first report to show that serum EV microRNA could be a prognostic biomarker for IPF.
The circulating microRNAs in the whole serum have been investigated by many researchers in their efforts to identify useful candidate biomarkers for various diseases [
28,
29]. However, previous reports and the data from our pilot study (Additional file
1: Figure S1) showed that analysing the microRNAs from the EV-rich serum fraction improved the reproducibility compared to the analysis of whole serum [
24,
25]. Furthermore, there is no clear consensus in the research community as to the appropriate normalization control for microRNA expression profiling in serum samples. Moreover, because the components of the EVs and their secretion dynamics vary according to their cellular origin and environment [
30], it is possible that the data obtained from an EV analysis would reflect the condition of chronic diseases, including fibrosis. Therefore, we focused on the EV-associated microRNAs in the serum and examined their changes during lung fibrosis. We hypothesized that the mouse model could be suitable for a comprehensive assay using PCR arrays due to its consistency and simple background relative to human subjects, although the mouse model does not ideally mimic the human disease. Therefore, we first analysed the changes in serum EV microRNAs in a mouse model of bleomycin-induced lung fibrosis to identify candidate microRNAs for biomarkers of fibrotic lung diseases. The PCR array assays identified the EV microRNAs that were changed in the mouse model. Among these microRNAs, additional confirmatory quantitative PCR assays revealed that miR-21-5p was upregulated in both the acute and chronic/fibrotic phase, which suggested that only serum EV miR-21-5p was changed throughout lung fibrosis and could serve as a biomarker for human fibrotic lung diseases, including IPF. In the human subjects, we examined the levels of miR-21-5p in the serum EVs and the levels of EVs in serum samples. Our data showed that, although there were no significant differences among healthy controls, COPD patients and IPF patients, the EV levels in serum were highly variable, even within the same subject group. We assumed that the levels of EVs would influence the levels of the microRNAs in the samples. Moreover, we were interested in the net change in the microRNA content of the EVs, rather than the total amount of the microRNA in the serum EV samples, because the change in the content of individual species, rather than the total content, reflects the condition of cells or tissues during disease. Therefore, we then attempted to normalize the expression levels of the serum EV miR-21-5p by dividing them by the EV amount in each serum sample (miR-21-5p copy number/signal intensity for CD9-positive pan-EVs). This normalization step also improved the variability in the measurements in the healthy control group. On the basis of these results, we used the normalized levels of serum EV miR-21-5p for our initial analyses.
The correlation analyses between the serum EV miR-21-5p and clinical parameters indicated that both normalized and non-normalized serum EV miR-21-5p levels were correlated with the rate of decline in the percent-predicted VC over 6 months. Furthermore, we demonstrated that the miR-21-5p baseline levels could predict the mortality of IPF patients during the 30-month follow-up period. Our data suggested that the levels of serum EV miR-21-5p at baseline are predictive of long-term mortality in IPF patients and can also predict the short-term disease progression in terms of the decline in lung function.
Two recent reports have analysed the expression of microRNAs in the serum samples of IPF patients [
31,
32]. These two reports showed that the miR-21-5p in whole serum was increased in the IPF patients compared to the healthy controls, which is consistent with our findings from the serum EV microRNA analyses. However, in our prospective cohort study, we first clearly demonstrated that the baseline levels of serum EV miR-21-5p were significantly correlated with the disease progression (a decrease in predicted % VC) and were associated with mortality during the 30-month follow-up period. Our study is the first report to suggest that the microRNA in the serum EVs could be a promising candidate for a prognostic biomarker in patients with IPF.
Our study has not elucidated the mechanism for the increase in the serum EV miR-21-5p in IPF patients. Accumulating evidence has suggested that miR-21-5p plays a vital role in various biological responses and pathological processes [
33‐
35]. A study that used miR-21 gene-targeted mice clearly showed that miR-21-5p targeted tumour suppressor genes, including
spry1,
pten, and
pdcd4 [
33]. The expression of miR-21-5p is also regulated at the post-transcriptional level by the TGF-β family of proteins and their downstream signal transducers, the SMADs [
36], which are key regulators in the pathogenesis of fibrosis. Our previous reports and those of others have shown that miR-21-5p is increased in whole-lung samples from both bleomycin-induced mouse models of lung fibrosis and human patients with IPF [
19,
37‐
39]. MiR-21-5p targets an inhibitory SMAD called SMAD7, and administration of miR-21-5p antisense probes attenuates the severity of the bleomycin-induced fibrosis by blocking the positive feedback loop of TGF-β signalling [
38]. MiR-21-5p is also expressed in lung epithelial cells, and increased miR-21-5p expression was observed in the lungs of patients with IPF [
19]. It is possible that the increased levels of serum EV miR-21-5p reflect the conditions of fibrotic lung diseases, including IPF, in which TGF-β signalling is one of the most relevant signalling pathways. However, miR-21-5p expression is also regulated by other factors that are involved not only in fibrosis but also in cell proliferation and inflammation. It is, therefore, likely that the reason for the increases in the levels of serum EV miR-21-5p in IPF patients is more complicated.
There are limitations to this study. First, the sample size for the human study was relatively small. This may explain why this study did not show that factors, including male gender, were risk factors for death, although these factors have been reported as independent risk factors for disease progression [
40,
41]. To confirm our observation, another cohort study with a larger sample size would be needed. Next, we isolated (or concentrated) the EVs from the sera using a reagent (Total Exosome Isolation reagents) and then examined both the amount of EVs using Exoscreen and the microRNA expression profiles, followed by the analyses of the microRNA expression profiles after normalization to the amount of EVs. However, the isolation reagent contains polymers that sequester the water molecules to force the less-soluble components, such as vesicles, to be precipitated. It is, therefore, possible that other vesicles, particles and protein aggregates could have contaminated the precipitate that contains the EVs. This isolation reagent may therefore not be the ideal method for the isolation of pure EVs. Exosomal biomarkers have the advantage of being more specific and stable compared to other biomarkers from the biological fluid. However, it will be very important to increase the efforts to establish a standardized method for the isolation of pure EVs and the microRNA inside these EVs for both disease prediction and pathogenesis elucidation. Third, Exoscreen seems to be a better available method to evaluate the amount of EVs on the basis that we observed that the total yield of RNA (mainly, small RNAs; Additional file
1: Figure S3) isolated from the serum EVs showed a good correlation with the relative levels of serum CD9-positive EVs (signal intensities (SIs) for CD9 positive EVs). However, it is possible that the EVs for which the expression is low or is downregulated in the pathological conditions may cause discrepancy between signal intensities (SIs) for CD9 positive EVs and the actual amount of EVs. Because there is no established method to count the absolute number of EVs or measure the absolute amount of EVs, it is difficult to solve this possible discrepancy at the present. We also think that it is also important to establish the method to measure the absolute number or amount of EVs in biological fluid samples for normalization. Moreover, to explore the method to count and isolate the specific EVs originated form specific types of cells (for example, epithelium or endothelium) or the cells under pathological conditions is thought to be useful for both establishing biomarkers and understanding pathogenesis of various diseases. Final, we first used the normalized levels of serum EV miR-21-5p for our analyses because we were interested in the net change in the microRNA contents of the EVs. However, although this normalization step improved the variability in the measurements in the healthy control group and some of the analysed results suggested that the normalization might have improved the ability to predict the mortality of the IPF patients (Additional file
1: Figure S7), it is also true that the benefit of this normalization seems to have been minimal in this study (Additional file
1: Table S8 and S9). To confirm the benefit of the normalization, another cohort study with a larger sample size and improvements in both the EV isolation and EV measurement would be needed.
Acknowledgements
We thank the Biomedical Research Core of Tohoku University Graduate School of Medicine for technical support. We very much appreciate Mr. Brent K. Bell for critical reading of the manuscript. This manuscript also has been edited in English by NPG Language Editing. The authors also thank the patients, healthy volunteers and participating staff members at our study sites.