Background
Over the past decade, the incidence of idiopathic pulmonary fibrosis (IPF) is gradually increasing globally [
1]. IPF generally affects men after the age of 60 years, and the prognosis of patients with IPF is extremely poor, with a median survival time of 2–4 years, even worse than many cancers [
2,
3]. Pathologically, IPF is characterized as a chronic, progressive fibrotic parenchymal lung disorder with excessive deposition of extracellular matrix (ECM) [
4]. Accumulating evidences suggest that plenty of mechanisms contribute to pathological change of IPF, such as epithelial injury and activation, inflammation, and abnormal remodeling [
4‐
7]. Despite great achievements have been made in study of pathological changes in IPF, little is known about the origin of activation of fibroblasts during fibrotic remodeling. A better understanding of the mechanism is greatly needed.
Non-protein coding RNAs (ncRNAs) account for the majority of RNA, and only approximately 1.9% of RNAs encode proteins [
8,
9]. Previously, ncRNAs were recognized as “evolutionary junk,” without any biological functions. However, with the development of the deep sequencing technology, accumulating evidences indicate that ncRNAs have great impacts on molecular mechanisms of animals and even human [
10‐
12]. Generally, ncRNAs can be divided into two categories based on size, and longer than 200 nucleotides (nt) are classified as long non-coding RNAs (lncRNAs), whereas smaller than 200 nucleotides are known as short non-coding RNAs, e.g. microRNAs (miRNAs), repetitive RNAs and intronic RNAs [
13,
14]. Among short non-coding RNAs, miRNAs, with a size of ∼20 nt, have attracted widespread attention [
15]. Reports have demonstrated that several lncRNAs and miRNAs play critical roles in the progression of IPF. For instance, Savary et al. reported that lncRNA DNM3OS played an important role in TGF-β-induced lung myofibroblast activation by regulating several miRNAs [
16]. Another lncRNA pulmonary fibrosis-associated (PFAL) was highly expressed in lung fibrosis tissue of experimental mice. In TGF-β1-induced fibrotic lung fibroblasts, PFAL could promote cell proliferation, migration, and fibroblast-myofibroblast transition by regulating miR-18a [
17]. In addition, lncRNA H19 [
18], MEG3 [
19] have been also confirmed to paly critical roles in IPF.
Previously, a large number of studies have focused on a certain lncRNA called SNHG16, which was reported to promote epithelial-mesenchymal transition (EMT) of many type of cancer cells [
20,
21]. More importantly, it could also promote proliferation and migration of lung cancer cells [
22]. However, whether SNHG16 plays regulatory roles in lung fibrosis remains unknown and still needs to be explored.
In the present study, we used both BLM-induced pulmonary fibrotic animal model and TGF-β1-induced pulmonary fibrosis in vitro model to explore the role of lncRNA SNHG16 in the progression of pulmonary fibrosis. The aim of this study was to clarify the regulatory role of SNHG16 and mechanisms of its down-stream factors in pulmonary fibrosis. Our study not only sets a novel sight into pathophysiological mechanism of IPF, but also provides a theoretical basis for the research of new therapeutic target for IPF.
Methods
Experimental pulmonary fibrosis model
All the animal-related experiments were performed based on the Ethics Committees of Shanghai Pudong New Area Gongli Hospital. C57BL/6 mice (male, 6–8 weeks old) were obtained from Charles River Animal Technology (Beijing, China). To establish the pulmonary fibrosis model, bleomycin (BLM, Sigma-Aldrich, Billerica, MA, USA) dissolved in saline was intratracheally administered (1.5 U/kg of body weight) under anesthesia. Equal volume of sterile saline was injected in mice which were used for control. In another series of experiments, 72 h after administration of BLM, mice were intratracheally injected with adenovirus-associated short hairpin RNA (shRNA) (VectorBuilder, Guangzhou, China) for lncRNA SNHG16 (Ad-sh-SNHG16#1, Ad-sh-SNHG16#2) or its control (Ad-sh-NC). Mice were sacrificed on the 28th day after BLM administration.
Histologic experiments
The pulmonary tissues were fixed in 4% paraformaldehyde solution for 7 days. After dehydration, tissues were embedded in paraffin for histopathological analysis. Sections were prepared with a thickness of 4 μm and stained with hematoxylin and eosin (H&E) or Masson’s trichrome kit (Shanghai, China) based on manufacturers’ instructions.
Immunohistochemistry (IHC)
Briefly, paraffin sections were treated with 0.3% H2O2 in methanol. Afterwards, sections were incubated with primary antibody Anti-α-SMA (1:1000, #ab32575, Abcam, Cambridge, MA, UK) overnight at 4 °C. The following day, goat anti-rabbit HRP labeled secondary antibody was added, and then immunostained using DAB plus kit. Images were acquired under a fluorescence microscope (Leica, Germany).
Western blotting
Proteins from different groups of lung tissues or fibroblasts were extracted using RIPA lysis buffer (Sigma, USA) and qualified by a BCA kit (Beyotime Biotechnology, Nanjing, China). Then, samples were separated by electrophoresis, transferred onto a PVDF membrane (Invitrogen, USA). After incubated in 5% blocking buffer, membranes were incubated with primary antibodies at 4 °C overnight: α-SMA (1:500, #ab7817, Abcam), E-cadherin (1:1000, # ab40772, Abcam), collagen 1 (1:1000, # ab34710, Abcam), fibronectin 1 (1:1000, # ab2413, Abcam), Notch2 (1:1000, # ab8926, Abcam), GADPH (1:1000, # ab8245, Abcam). The next day, secondary antibodies were added and incubated for 40 min. Images were detected using the Odyssey Infrared Imaging System.
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from lung tissues or cultural lung fibroblasts using TRIzol reagent (Invitrogen, USA). cDNA was synthesized by reverse transcriptase kit (Takara, Otsu, Japan). Gene expression was determined with SYBR Green I on a ABI 7500 fast Real-Time PCR instrument (California, USA). The primer sequences for SNHG16, GAPDH, miR-455-3p, U6 were listed in Table
1. Relative mRNA levels and miRNA were calculated based on the Ct values and normalized to the GAPDH or U6 expression in each sample, respectively. Data were analyzed using the 2
−ΔΔCT method.
Table 1
Primers used in the present study
miR-455-3p | forward: 5′-ATAAAGRGCRGACAGTGCAGATAGTG-3′ |
reverse: 5′-TCAAGTACCCACAGTGCGGT-3′ |
U6 | forward: 5′-GCTTCGGCAGCACATATACT-3′ |
| reverse: 5′-GTGCAGGGTCCGAGGTATTC-3′ |
SNHG16 | forward: 5′-CCAAGCTTATGCCAGATGGGATCAGCAC-3′ |
| reverse: 5′-CCGCTCGAGCTTGGTGAGTCAACACTGGGT-3′ |
GAPDH | forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′ |
Cell culture and treatment
Primary mouse lung fibroblasts were isolated from 2-day-old C57BL/6 mice. The lung tissue was removed and digested with 0.25% trypsin. Isolated cells were washed and resuspended in DMEM (Gibco, Grant Island, NY, USA) supplemented 10% fetal bovine serum (Gibco, USA), and 1% penicillin G/streptomycin (Beyotime Institute of Biotechnology, Nanjing, China). Lung fibroblasts were cultured at 37 °C with 5% CO2. For treatment, recombinant human TGF-β1 (10 ng/ml; PeproTech, USA) was used for establishing pulmonary fibrosis model in vitro.
Cell transfection
Prior to TGF-β1 treatment, lung fibroblasts were treated with serum-free culture medium for 5 h. Then, lentivirus-mediated short hairpin RNA (shRNA) target SNHG16 or its negative control (sh-NC), miR-455-3p mimics or inhibitor and their control plasmids (NC mimic, NC inh), pcDNA3.1( +) Notch2 Vector for overexpressing Notch2 and its control (mock) purchased from GenePharma (Shanghai, China) were transfected into lung fibroblasts using lipotectamine 2000 (Invitrogen, Carlsbad, CA, USA) based on manufacturer’s instructions.
Immunofluorescence staining
Transfected lung fibroblasts were fixed with 4% paraformaldehyde solution. After permeabilization and blocking treatment, cells were incubated with primary antibody against mouse α-ASM (1:500, # ab83354, Abcam) overnight at 4 °C. Next, sections were incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse in dark. Finally, cells were stained with diamidino-2-phenylindole (DAPI; 1:1000; Beyotime, Nanjing, China) for 15 min. Data were acquired and analyzed using a fluorescence microscope (Nikon 80i, Tokyo, Japan).
Cell proliferation assays
EdU staining was used for detecting cellular proliferation. Briefly, lung fibroblasts were cultured in 24-well plates. Cell-Light EdU DNA Cell Proliferation Kit (RiboBio, Guangzhou, China) were used for detecting cell proliferation based on manufacturer’s instructions.
Wound-healing assay
Different groups of transfected lung fibroblast cells were seeded into 6-well culture plates and grew until ~ 80% confluence. The cell monolayer was wounded by scratching with a 200 µl pipette tip. The movement of cells was calculated using an olympus microscope at 0 and 24 h.
Dual-luciferase reporter assay
Putative wild-type (WT) and mutant (Mut) miR-455-3p binding sites in the 3ʹ-UTR of SNHG16 (SNHG16-WT, SNHG16-Mut) and Notch2 (Notch2-WT, Notch2-Mut) were cloned into a pmirGLO-Report luciferase vector (Promega, Madison, WI, USA). HEK293 cells were transfected with SNHG16-WT or SNHG16-Mut, as well as Notch2 3′UTR WT or Notch2 3′UTR Mut, followed transfecting with miR-455-3p mimics or NC mimics using Lipofectamine 3000 (Invitrogen, USA). An amount of 5 ng/well Renilla luciferase plasmid was used as internal control. Luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, USA) 48 h after transfection.
RNA binding protein immunoprecipitation (RIP) assay
The RIP assay was used to explore the binding relationship between endogenous SNHG16 and miR-455-3p. Briefly, lysate of transfected lung fibroblasts was treated with RIP buffer containing magnetic beads conjugated with anti-Ago2 antibody (Millipore, Billerica, MA, USA), or negative control IgG. Wash buffer was used to wash the beads, and the complexes were incubated with 0.1% SDS/proteinase K to remove proteins. qRT-PCR assay was performed thereafter.
Statistical analysis
All experiments were repeated in triplicate. Data were exhibited as mean ± SD. A one-way ANOVA followed by Dunnett’s test was used for multiple comparisons. A two-tailed p value less than 0.05 was considered as statistically significant difference. Statistical analyses were carried out using the GraphPad Prism 7.0 and Statistical Package for Social Sciences version 21.0 (SPSS, Inc., Chicago, IL).
Discussion
With the rapid development of deep transcriptome sequencing technology, research on non-coding RNAs has grown increased exponentially [
23]. Although in some cases, without protein coding ability, ncRNAs seem lack of bio-function, more and more evidence confirmed that they play critical roles in controlling gene expression through a variety of mechanisms, e.g. targeting transcripts [
24], affecting splicing function [
25], targeting cis-acting promoter RNAs [
26]. In our study, we found that lncRNA SNHG16 was highly expressed in BLM-induced pulmonary fibrotic animal tissues. Moreover, lung fibrogenesis can be affected by expression of SNHG16. We also explored the potential mechanism of how SNHG16 exerted its role, and we found that SNHG16 directly targeted miR-455-3p, thereby decreasing protein levels of Notch2. Our data indicated that lncRNA SNHG16 could promote pulmonary fibrosis by targeting miR-455-3p to regulate the Notch2 pathway.
Regarding the pathogenesis of IPF, repeated micro-injury of the alveolar epithelium over time is considered to be the first trigger of the maladaptive repair process [
27]. Subsequently, aberrant epithelial-mesenchymal crosstalk leads to an imbalance between profibrotic and antifibrotic mediators, followed by fibroblasts, myofibroblasts proliferation and accumulation of extracellular matrix [
28,
29]. In these process, epithelial mesenchymal transition (EMT) has been considered to be the milestones of fibrogenesis [
30]. Previous studies confirmed that lncRNA SNHG16 acts as a promotor of EMT process in variety of cancer types, e.g. glioma [
31], esophagus cancer [
21], cervical cancer [
32], and gastric cancer [
20]. Therefore, our hypothesis was that lncRNA SNHG16 may also play important roles in lung fibrogenesis. To this end, we not only used BLM-induced pulmonary fibrotic animal model and TGF-β1-induced pulmonary fibrotic cell model in vitro, but also employed different series of transfected lung fibroblast cells systems to explore the underlying molecular mechanisms. Results demonstrated that knockdown of SNHG16 could attenuate BLM-induced pulmonary fibrosis, and suppress the proliferation and migration process of lung fibroblast cells. These findings suggested that SNHG16 palys an accelerator of lung fibrogenesis which is in line with previous studies.
Increasing studies have reported that, in some cases, lncRNA and miRNA can cross-talk in a competing endogenous RNA (ceRNA) process [
33]. In this condition, lncRNAs might act as “sponge”, thus, miRNAs are isolated from their targeted mRNAs, leading to the expression changes of their target genes [
34]. In our study, by using dual-luciferase reporter assay and RIP assay, we found that SNHG16 was able to bind to miR-455-3p. Moreover, the expression of miR-455-3p can be regulated by SNHG16. Furthermore, Notch2 was confirmed to be a target of miR-455-3p, and can be regulated by both SNHG16 and miR-455-3p to affect the process of lung fibrogenesis.
As important candidates in our study, miR-455-3p and Notch2 have also been confirmed to be involved in pathological progressions of lung fibrogenesis in many published studies. Previous study showed that miR-455-3p could act as a fibrogenesis suppressor. It may exert its role by potentially binding to Bax gene to suppress apoptosis of alveolar epithelial cells [
35]. Notch2 is one of the most important members of Notch receptor family, composed of 34 exons and encoded by 2471 amino acids [
36]. Studies have demonstrated that Notch2 can be detected in bronchiolar epithelial cells [
37], and plays important roles in small-cell lung cancer [
38]. In IPF study field, Notch2 was reported to be involved in immune system [
39]. More evidence regarding mechanisms of Notch2 in IPF are greatly needed to valid the current results.
Conclusions
Taken together, the results from this study demonstrated that lncRNA SNHG16 served as an essential regulator in lung fibrosis. Moreover, SNHG16 functioned as a miR-455-3p sponge, thereby positively regulating Notch2 expression by binding miR-455-3p. Our findings might provide a novel insight into pathological of fibrosis. However, there are remaining puzzles need to be solved, such as whether the expression of SNHG16 is related with prognosis of patients with IPF, and how SNHG16 exerts its role in different stage of IPF. Therefore, in future, human-related clinical studies will be required.
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