Background
Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic and progressive fibrosing interstitial pneumonia of unknown cause, and this condition occurs primarily in adults and often develops in the lungs [
1]. The incidence of IPF increases remarkably with age; most patients with IPF are older than 60 years at the time of diagnosis, and young individuals are seldom affected by IPF [
2]. Thus, a mechanistic link possibly exists between chronological age and this disease, although the relationship between them remains uncertain. Oxidative stress disrupts the balance between oxidant production and antioxidant defense mechanisms in tissues, and this process plays a major role in the pathogenesis of aging [
3]. López-Otín et al. proposed several pivotal hallmarks, such as abnormal shortening of telomeres, epigenetic change, and mitochondrial dysfunction, which contribute to the aging process. These hallmarks can occur simultaneously and become interconnected during aging [
4]. Thus, the mechanistic link among these aging hallmarks in IPF should be investigated.
Long noncoding RNAs (lncRNAs) are distinct fields of imprint in gene dosage compensation and considered by biochemists, geneticists, and computational biologists as an underlying factor in epigenetic regulation [
5]. The first identified lncRNA is a gene associated with lung adenocarcinoma and designated as metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) in respiratory diseases [
6]. Studies on gene knockdown have shown that MALAT1 regulates the expression of genes, particularly those that do not undergo splicing and are involved in cell migration, colony formation, and metastasis [
7,
8]. Our laboratory studies initially demonstrated the relationship among lncRNAs, as well as their adjacent or homologous protein-coding genes and putative miRNA-target sites. On the basis of our results, lncRNA transcription was proposed to affect the expression levels of genes adjacent to
cis elements, hybridize to the overlapping sense transcript, act as a ceRNA, or regulate gene expression in pulmonary fibrosis [
9‐
11]. Huang et al. [
12] also supported our findings by performing motif search and manual comparisons of the data reported regarding the lungs of patients with IPF. Nevertheless, the mechanistic link between aging and lncRNA in the pathogenesis of IPF are rarely investigated.
Telomeric repeat-containing RNA (TERRA) is a type of lncRNA. Telomeres are transcribed from subtelomeric regions to large noncoding TERRA by RNA polymerase II. In the absence of regulatory mechanisms, the chromosome ends of telomeres deteriorate; these telomeres consequently become dysfunctional and thus promote DNA damage, which causes cellular senescence or rampant genome instability and apoptosis [
13]. TERRA overexpression in single telomere can induce early-onset senescence [
14,
15]. Telomerase activity is detected in some highly proliferative tissues, but the amount of telomerase expressed in human somatic cells is insufficient to trigger replicative senescence upon a defined number of cell divisions caused by telomere erosion. Telomerase inactivation acts as a tumor-suppressing mechanism when its re-expression in human fibroblasts facilitates the bypassing of senescence. As a result, cellular immortalization occurs. Accordingly, 90% of human cancers reactivate telomerase activity, which stabilizes telomere length [
16].
Reactive oxygen species (ROS) elicit adverse effects on lung epithelial cells and fibroblasts. Fitch et al. [
17] reported that lung epithelial SHH in patients with IPF increases under oxidative stress. Fernandez et al. [
18] reported that ROS or hyperoxia-activated damage is an initial defense mechanism and harmful environmental stimulus in homeostatic regulation of the lung microenvironment in IPF. They also found that decreasing the oxidative load in lungs can be therapeutically beneficial. Mitochondria are well-known organelles that produce ROS and frequently function as a source of oxidants associated with oxidative stress. In the present study, the regulation of TERRA on telomeres and mitochondria in human alveolar epithelial cells of IPF was investigated. Overall, our findings may provide a diagnostic and therapeutic target to ameliorate age-associated pathologies and improve the health of patients with IPF.
Methods
IPF patients
IPF was diagnosed in accordance with the American Thoracic Society/European Respiratory Society consensus criteria [
1], which include clinical, radiographic, and characteristic histopathological features (
n = 24). Blood sample (5 mL) was drawn from each participant and prepared for testing. Healthy persons (
n = 24) whose age and gender corresponded to those of the patients with IPF were then selected. Each participant provided a written informed consent. The local ethics committee approved this study.
Animal model
C57BL/6 mice (8 weeks old) were obtained from the Model Animal Research Center of Nanjing University (Nanjing, China). All of the animal experiments were performed on the basis of the regulations established by the Ethics Committee on the Animal Experiments of Binzhou Medical University (196,738, 01/09/2014) [
11]. The mice were housed under a 12 h light/dark cycle and allowed free access to food and water. The mice were then randomly divided into two groups with 10 mice in each group: sham group and bleomycin (BLM)-treated group. On day 28, all of the mice were killed, and lung tissue sections were collected and immediately frozen in liquid nitrogen for further analysis. The BLM animal model was administered with 5 mg/kg BLM dissolved in saline through single intratracheal instillation under anesthesia as previously described [
9,
10].
Cell culture
A549 (human type II alveolar epithelial cell) and MLE-12 (mouse type II alveolar epithelial cell) were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). These cells were maintained in Dulbecco’s modified Eagle’s medium for A549 and advanced minimum essential medium for MLE-12 respectively, and supplemented with 2 mM l-glutamine, 10% heat-inactivated FBS (Gibco, USA), 100 U/mL penicillin, and 100 mg/mL streptomycin.
Cell proliferation assay
Cell proliferation was determined using a cell counting kit-8 (CCK-8; Beyotime Inst Biotech, China) in accordance with the manufacturer’s instructions. In brief, cells were seeded in a 96-well flat-bottomed plate at 1 × 106 cells/well and then grown at 37 °C for 24 h. After 10 μL of WST-8 dye was added to each well, the cells were incubated at 37 °C for 2 h. Absorbance was determined at 450 nm by using a microplate reader. Cell proliferation was calculated on the basis of the coloration depth by using the following formula: cell proliferation (%) = (measurement tube absorbance − absorbance of blank) / (standard pipe of absorbance − absorbance of blank) × 100%.
Cell growth curve analysis
An xCELLigence real-time cell analyzer (ACEA Biosciences, Inc., Hangzhou, China) was placed in an incubator in advance. Afterward, 1 × 105/mL cells were seeded in a test E-Plate and placed in an analyzer, which can automatically record the cell growth curve.
Transmission electron microscopy (TEM) observation
Cells or lung tissues were fixed with 3% fresh glutaraldehyde at 4 °C for at least 4 h and then postfixed in 1% osmium tetroxide for 1.5 h. The treated samples were dehydrated in gradient ethanol, infiltrated with Epon812, and embedded. The tissues were cultured at 37, 45, and 60 °C for 24 h. Ultrathin sections were prepared using an Ultracut E ultramicrotome. The resulting sections were stained with uranyl acetate and lead citrate and observed under a JEM-1400 TEM system (JEOL Ltd., Tokyo, Japan), as previously described [
19].
Mitochondrial membrane potential (MMP) assay
MMP was determined using 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimi dazolycarbocyanine iodide (JC-1; Beyotime Biotechnology, China) stain in accordance with the manufacturer’s instructions. Briefly, the harvested cells were resuspended in a mixture containing 500 μL of culture medium and 500 μL of JC-1 (5 μg/mL) staining fluid. The resulting mixture was incubated in the dark at 37 °C for 20 min. After the cells were washed twice with ice-cold staining buffer through centrifugation, the cells were resuspended in 500 μL of culture medium and then analyzed through flow cytometry. The values of MMP staining for each sample were expressed as red and green fluorescence intensities.
Caspase activity
Caspase-3 and caspase-9 activities were obtained on the basis of the cleavage of chromogenic caspase substrates acetyl-Asp-Glu-Val-Asp
p-nitroanilide (Ac-DEVD-pNA) and acetyl-Leu-Glu-His-Asp
p-nitroanilide (Ac-LEHD-pNA) by using an assay kit (Beyotime Biotechnology), as previously described [
20]. The cell lysate from 1 × 10
6 cells was incubated at 37 °C for 2 h with 200 μM of Ac-DEVD-pNA (caspase-3 substrate) or Ac-LEHD-pNA (caspase-9 substrate).
The absorbance of the yellow pNA cleavage from its corresponding precursors was determined by using a spectrometer at 405 nm in a microplate reader (SpectraMax M2). The total protein concentrations in the supernatants were measured through the Bradford method.
Apoptosis assay by flow cytometry
Suspended and adherent-treated cells (1 × 106) were collected and washed with cold PBS. The fixation fluid was also washed with PBS, and 500 μL of binding buffer was added to resuspend the cells. Afterward, 5 μL of Annexin V-FITC and 5 μL of propidium iodide staining solutions were added for 20 min in the dark. Apoptosis rate was identified through flow cytometry (Beckman, Fullerton, CA, USA).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the blood samples/cells of the IPF patients by using TRIzol reagent from Invitrogen (Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. Complementary DNA was synthesized by using M-MLV reverse transcriptase kit from Promega (Madison, WI, USA) in accordance with the manufacturer’s instructions. qRT-PCR was performed using a SYBR Green PCR Master Mix kit from Takara Bio (Shiga, Japan) on a Rotor Gene3000 real-time PCR system from Corbett Research (Sydney, Australia). The following PCR conditions were set: initial denaturation at 95 °C for 5 min, 30 cycles at 60 °C for 25 s, annealing at 52 °C for 20 s, and extension at 72 °C for 20 s. Fluorescence signal was monitored at 585 nm during each extension. Glyceraldehyde 3-phosphate dehydrogenase served as an internal control.
ROS generation assay
Intracellular ROS levels were estimated using a kit containing the membrane-permeable fluorescent probe 2,7-dichlorofluorescin diacetate (Beyotime Biotechnology). The cells were co-activated with 5 mM of 2,7-dichlorofluorescin diacetate for 30 min. Colorimetric intensity was determined using a fluorescence spectrophotometer at excitation of 484 nm and emission of 531 nm.
Superoxide dismutase and catalase activities
Superoxide dismutase (SOD) and catalase activities in the A549 cells were respectively determined with a total SOD assay kit and a catalase assay kit (Beyotime Biotechnology, China) following the manufacturer’s protocol [
20].
Transfection of siRNA-TERRA
The siRNAs used in our experiment were synthesized by RiboBio Co., Ltd. (Guangzhou, China). Two siRNA fragments were designed and synthesized. Afterward, 1 × 105 cells were seeded in 24-well plates and cultivated with a 1640 medium containing 10% newborn calf serum for 24 h. Subsequently, 1.25 μL of 20 μM siRNA was diluted with 50 μL of 1× riboFECT™ CP buffer, and the resulting solution was incubated for 5 min at room temperature. Approximately 5 μL of riboFECT™ CP reagent was added to the solution, which was then incubated for 15 min at room temperature. The mixed solution was added to 443.75 μL of 1640 medium without 10% newborn calf serum. The cells were incubated with the mixed solution for 48 h.
Real-time fluorescent quantitative TRAP assay
Telomerase activity was detected with a fluorescent quantitative TRAP (FQ-TRAP) kit in accordance with the manufacturer’s protocol. In brief, 1 × 106 cells were lysed in 50 μL of Reagent A containing RNase inhibitors and incubated at 4 °C for 30 min. The lysate was then centrifuged at 12,000×g for 30 min at 4 °C, and the supernatant was collected. Protein concentration was obtained using the Bio-Rad protein reagent set. The total volume of the reaction mixture was 25 μL, which contained 15 μL of Reagent B, 1 μL of cell lysis solution, 2.5 μL of Reagent C, and 6.5 μL of Reagent D. PCR was initiated at 30 °C for 20 min, followed by 29-cycle amplification (95 °C for 20 s, 50 °C for 30 s, and 72 °C for 90 s), and terminated at 60 °C for 90 s. The telomerase activities in the cells were determined on the basis of the threshold cycle.
Western blot analysis
The cells were collected and lysed in SDS sample buffer with protease inhibitors. Protein concentrations were determined through BCA method. First, 20 μg of proteins were separated through SDS-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane blocked with bovine serum albumin (5%) in TBS-T. Afterward, the membrane was incubated with anti-proliferating cell nuclear antigen (anti-PCNA), anti-telomerase reverse transcriptase (anti-TERT), and anti-GAPDH antibodies at 4 °C overnight. The blots were then treated with an HRP-labeled goat antirabbit IgG (1:5000; Beijing Zhong Shan-Golden Bridge Technology Co., Ltd., Beijing, China) for 1.5 h. The membranes were subsequently washed with TBST and incubated with ECL reagent before exposure.
Telomere length assay
After extraction of the whole genome DNA by QuickGene 610 L automatic extractor, the first qRT-PCR test was performed. The detection of telomere length primer was as follows: SEQ ID NO.1: CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT; SEQ ID NO.2: GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT. The reaction system was: 4.3 μL 2× SuperReal Premix Plus, 0.3 μL SEQ ID NO.1 (10 μM), 0.3 μL SEQ ID NO.2 (10 μM), 5 μL DNA (1 ng/μL to 5 ng/μL). The total reaction volume was 10 μL. The following PCR conditions were set: initial denaturation at 98 °C for 10 min, 45 cycles at 95 °C for 30 s, and 60 °C for 1 min. Then, the second qRT-PCR test was performed. The reaction system was: 4.7 μL 2 × Taqman Expression Master Mix, 0.3 μL RNase P, 5 μL DNA (5 ng/μL). The total reaction volume was 10 μL. The following PCR conditions were set: initial denaturation at 98 °C for 10 min, 45 cycles at 95 °C for 30 s, and 60 °C for 1 min. Relative telomere length = Telomere repeat copy number/Single-gene copy number.
Statistical evaluation
Data were statistically analyzed in SPSS version 19.0 (IBM SPSS Statistics Company, Chicago, USA). Data were presented as mean ± standard deviation of at least three independent experiments. Unpaired Student’s t test was used to compare the two groups. One-way ANOVA with Student–Newman–Keuls post hoc test was conducted to compare three or more groups. Statistical significance was considered at P < 0.05.
Discussion
Many advances enhanced our understanding of IPF pathogenesis and presented substantial evidence supporting several aging mechanisms, including oxidative stress, telomere length regulation, mitochondrial dysfunction, and changes in the number of anti-aging molecules in IPF [
27]. A further understanding of these abnormalities may help design and improve novel therapeutic interventions for IPF patients. In particular, lncRNA is considered essential for various molecular and cellular processes, such as senescence, proliferation, apoptosis, and differentiation. Therefore, the influence of lncRNA on cellular and molecular bases of aging was reviewed [
28]. However, the underlying mechanisms of lncRNAs in IPF remain poorly understood. The regulation of lncRNA and its interference should be investigated to explain the pathogenesis of IPF and provide a biomarker or therapeutic target for IPF patients. In the present work, the TERRA expression levels in the peripheral blood mononuclear cells from IPF patients were inversely correlated with FVC%. This finding is considerably important because lung biopsy is the only method that can accurately diagnose IPF. However, lung biopsy is an invasive procedure with high morbidity and mortality risks, and it may be unsuitable for some individuals. Determining an appropriate biomarker in the peripheral blood of IPF can provide a useful noninvasive method for IPF treatment. Thus, regulating the TERRA expression level can provide a novel biomarker or therapeutic approach for IPF treatment. In our study, a cell model was used to demonstrate that TERRA might contribute to the IPF pathogenesis, and RNA interference on TERRA can improve telomeric and mitochondrial functions (Fig.
7).
Oxidative stress plays a key role in aging because oxidative changes can provide mechanistic switches that control protein conformation, catalytic activity, protein–protein interaction, protein–DNA interaction, and protein trafficking [
29,
30]. IPF Patients possess increased markers of oxidative stress locally in their lungs and systemically in their bodies [
31,
32]. As a marker of oxidative stress, ROS promotes the occurrence of pulmonary fibrosis by activating the crucial mediators of epithelial–mesenchymal transition. Protection from free radicals is attained through different mechanisms, such as SOD and catalase, which are enzymatic systems that decompose superoxide radicals to H
2O
2. As expected, the increased ROS generation in the H
2O
2-induced lung fibrosis models was more evident than that in the other groups. siRNA-TERRA can also suppress ROS generation. Evidently, the activities of SOD and catalase decreased in the H
2O
2-induced lung fibrosis models, and siRNA-TERRA promoted SOD and catalase activities. Accordingly, the dysregulation of lncRNA expression plays a key role in cellular stress responses [
33,
34]. Our findings indicated that TERRA can facilitate IPF pathogenesis.
Telomeres protect cells, facilitate cell proliferation, and enable DNA polymerase to complete replication. The length of telomere corresponds to the length at birth and rate of attrition thereafter. The latter indicates several factors, such as cumulative oxidative stress [
35], which acts on progenitor cells. Thus, replicative senescence is elicited when the telomere length is decreased substantially. Accordingly, replicative senescence can be delayed by restoring telomerase expression, which replenishes telomeres. Critically short telomeres initiate the major senescence-regulatory pathway of p53; consequently, normal activity is reduced and age-related diseases are exacerbated [
36]. In particular, genetic and clinical evidence indicates that short telomere phenotype appears in most IPF patients [
37,
38]. Short telomeres in lung epithelial cells and peripheral blood cells are also observed in patients with IPF [
39]. Remarkably, 10% of patients with familial pulmonary fibrosis experience mutations in TERT, which is a key factor in telomere elongation [
40]. TERRAs also binds telomerase core components, TERC and TERT, but their role in telomerase function is unclear. On the one hand, TERRA can inhibit telomerase activity in vitro. On the other hand, in yeast, TERRAs are induced at short telomeres and form TERRA-TERC RNA clusters [
41]. Bleomycin causes an initial increase, and then a reduction, in telomerase activity controlled, at least in part, at the mRNA level. When telomerase activity is diminished, significant apoptosis of epithelial cells is initiated. With further disruption of telomerase activity, apoptosis occurs at significantly higher levels [
42].
We demonstrated that TERRA can inhibit telomerase activity and TERT expression to prevent telomere elongation. Consequently, TERRA can prevent the expression of PCNA, cyclin D1, and cyclin E. Our findings were consistent with previous results, which showed that telomeres are reduced during cell cycle when DNA polymerase is used because the priming of DNA synthesis does not readily occur in this region [
43,
44]. In our study, p53 was activated by H
2O
2, but its expression was inhibited when TERRA was interrupted by siRNA. On the basis of these results, we inferred that increased TERRA expression levels are associated with telomere dysfunctions caused by oxidative stress, and this phenomenon contributes to IPF pathogenesis.
Lungs are among the organs most exposed to various forms of ROS because of their high-oxygen-containing environment. The respiratory chain in the mitochondria is a significant endogenous source of ROS. Thus, the protective role of mitochondria in pulmonary fibrosis is closely associated with their function in ROS balance maintenance [
24]. Sosulski et al. [
45] proposed that enhancing the autophagic flux and mitochondrial recycling through hormetic compounds can diminish the expression of damaging ROS, maintain mitochondrial functions, normalize lung fibroblast phenotype, and promote a healthy lung. In the present study, the lung epithelial cells exhibited a substantial accumulation of dysmorphic and dysfunctional mitochondria, which supported our hypothesis. We also evaluated the cell apoptotic rate, changes in mitochondrial morphology, MMP, and electron transport chain. Dysfunctional mitochondria promoted cell apoptosis, caused structural integrity failure, decreased MMP, and damaged the electron transport chain under oxidative stress. These adverse conditions were mitigated to varying degrees when TERRA was inhibited by siRNA. Mitochondria are particularly susceptible to aging, and related abnormalities often include enlargement, loss of cristae, destruction of inner membranes, swelling, and impaired respiration. Bueno et al. [
46] evaluated the mitochondria in the lung epithelial cells of human patients with IPF and proposed that dysfunctional mitochondria promotes fibrosis in an aging lung.
Despite extensive research efforts over the past decades, effective therapies that uses corticosteroids, azathioprine, and cyclophosphamide for IPF are unavailable; thus, lung transplantation is currently the only effective therapy [
47,
48]. Alternative approaches and novel targets and pathways in IPF are urgently necessary to overcome poor prognosis and lack of available therapies. Although miR-122 is unrelated to IPF, targeting liver-specific miR-122 through an antisense-based approach is currently undergoing human trials to treat hepatitis C virus [
49]. Therefore, the cellular and molecular mechanisms of lncRNA and its antisense on IPF should be examined. Considering the trait of lncRNA, we should further explore new fields for IPF treatment.