Long non-coding RNA H19 deficiency ameliorates bleomycin-induced pulmonary inflammation and fibrosis

doi:10.21203/rs.3.rs-41983/v4

Download PDF

Research

Long non-coding RNA H19 deficiency ameliorates bleomycin-induced pulmonary inflammation and fibrosis

https://doi.org/10.21203/rs.3.rs-41983/v4

This work is licensed under a CC BY 4.0 License

Journal Publication

published 02 Nov, 2020

Read the published version in Respiratory Research →

You are reading this older preprint version

Read the latest preprint version →

Background: The poor understanding of pathogenesis in idiopathic pulmonary fibrosis (IPF) impaired development of effective therapeutic strategies. The aim of the current study is to investigate the roles of long non-coding RNA H19 (lncRNA H19) in the pulmonary inflammation and fibrosis of IPF.

Methods: Bleomycin was used to induce pulmonary inflammation and fibrosis in mice. The mRNAs and proteins expression in lung tissues was determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. H19 knockout (H19^-/-) mice were generated by CRISPR/Cas9.

Results: The expression of H19 mRNA was up-regulated in fibrotic lungs patients with IPF as well as in lungs tissues that obtained from bleomycin-treated mice. H19^-/- mice suppressed bleomycin-mediated pulmonary inflammation and inhibited the Il6/Stat3 signaling. H19 deficiency ameliorated bleomycin-induced pulmonary fibrosis and repressed the activation of TGF-β/Smad and S1pr2/Sphk2 in the lungs of bleomycin-treated mice.

Conclusions: Our data suggests that H19 is a profibrotic lncRNA and a potential therapeutic target for pulmonary fibrosis.

Pulmonology

Pulmonary Fibrosis

lncRNA H19

Smad

S1pr2

Idiopathic Pulmonary Fibrosis (IPF) is a progressive and highly lethal pulmonary fibrotic lung disease with poor treatment and unknown etiology, which rises significantly with age and higher in men [1-4]. Patients with IPF present similar characteristics to the usual interstitial pneumonia (UIP), including extracellular matrix deposition, alveolar architectural disruption, and subpleural honeycombing [5]. The patients with IPF usually have clinical experiences from cough to respiratory insufficiency and have a median survival time of 3 to 5 years after diagnosis [1, 4]. Unfortunately, there are currently no effective therapies capable of stabilizing or improving lung function for patients with IPF.

Long non-coding RNAs (lncRNAs) are defined as non-protein encoding RNA molecules that are more than 200 bp long in length [6]. lncRNAs have been shown to play important roles in different physiological activities, such as gene imprinting, cell proliferation, differentiation, apoptosis, migration, and immune responses [7, 8]. Recent studies have shown that aberrant expression of lncRNAs are associated with a number of human diseases, including cardiovascular, neurodegenerative, lung diseases, tumors and infections [9-15]. The lncRNA H19 is an imprinted and maternally expressed gene that plays a vital role in the controlling the cell proliferation and differentiation [16-18]. The others and our recent studies both indicate that hepatic H19 level is correlated with the severity of cholestatic injury and liver fibrosis in mice [19, 20]. Furthermore, H19 was also related to progression of lung cancer and lung fibrosis [21-24]. Although these studies suggest a causal link among H19 and pulmonary injury, it remains unknown whether and to what extent H19 is involved in the regulation of pulmonary fibrosis in vivo. In present study, we identified H19 as an up-regulated lncRNA in the lungs of pulmonary fibrosis. We further determined the functional roles and underlying mechanisms of H19 in pulmonary fibrosis, which suggested H19 acts as a profibrotic lncRNA in the lungs.

Materials

Hydroxyproline Assay Kit (Cat.No.MAK008-1KT, Sigma-Aldrich, St. Louis, MO), PowerUp SYBR-Green Master Mix kit (Cat. No. A25742) and a High Capacity cDNA Reverse Transcription kit (Cat. No. 4368814) were obtained from Applied Biosystems (Foster City, CA). NuPAGE 10% Bis-Tris gel (Cat.No.NP0301BOX, Invitrogen, Carlsbad, CA). Bleomycin (Cat.No. HY-17565, MedChemExpress, LLC, NJ), The antibodies used in this study were showed in Table S1.

A mouse model of bleomycin-induced pulmonary fibrosis

H19^-/- (H19 ΔExon1-5) mice were generated by CRISPR/Cas9-mediated genome engineering in C57BL/6J mice as our previously described [20]. The animal procedures were approved by the Shanghai Jiao tong University School of Medicine affiliated Xin Hua hospital Animal Care and Use Committee (XHEC-F-2020-008). The mice (about 8 weeks old) were divided into four groups: wild type (Wt) sham (n=8 -12), H19^-/- sham (n=6 -10), wild type (Wt) treated with bleomycin (BLM) (n=8 -12) and H19^-/- treated with bleomycin (BLM) (n=8 -12). For bleomycin administration, mice were anaesthetized with 2% isoflurane, and then instilled intratracheally with bleomycin (3.5 mg/kg body weight) in 100 ml PBS, as previously described [25, 26]. After 4 weeks, the lung tissues were collected for RNA, protein, collagen content analyses and histological analysis. The degree of fibrosis was quantitated using an Ashcroft score in a blinded manner according to the described method [27].

Fluorescence in situ hybridization (FISH)

H19 FISH in mouse lung tissue was performed using a commercially available RNAscope Multiplex Fluorescent Reagent Kit v2 (Advanced Cell Diagnostics, Newark, CA) and RNAscope® Probe-Mm-H19 (#423751, Advanced Cell Diagnostics, Newark, CA) according to the manufacturer’s instruction. Fluorescent staining targeting Sftpc protein was performed followed H19 FISH.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from left lung of mice using the RNeasy kit (Qiagen, Hilden, Germany) according to the protocol of the manufacture and 2 mg of total RNA was used to synthesize the 1st cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems, Foster City, CA). The real-time PCR reactions were performed on the ViiA 7 Real-Time PCR System (Applied Biosystems, Foster City, CA) using PowerUp SYBR-Green Master Mix kit (Applied Biosystems, Foster City, CA). All samples were assayed in triplicate, and data were normalized to endogenous control Hprt1. Relative RNA expression levels were calculated using the ^ΔΔCt method. The primers are listed in Table S2.

Western blotting

A total 30 mg left lung tissues was homogenized in 300 μl RIPA buffer (Invitrogen, Carlsbad, CA) supplemented with a protease inhibitor cocktail (Servicebio, Wuhan, China). After determining the protein concentration, the equal amounts of protein were separated on NuPAGE 10% Bis-Tris gels (Invitrogen, Carlsbad, CA) and transferred onto polyvinylidene diﬂuoride (PVDF) membranes. After blocking in 5% nonfat milk at room temperature for 60 min, membranes were incubated with the primary antibodies overnight at 4°C.The membranes were washed three times for 30 min with TBST (containing 0.1% Tween-20), and then incubated with secondary antibodies. After ﬁnal washes with TBST, the signals were detected using ECL chemiluminescence reagent Kit (Pierce, Rockford, IL, USA). The primary antibodies performed in this study as showed in Table S1.

Histology and immunofluorescence (IF)

The right lung tissues were immediately fixed in 10% neutral buffered formalin for 24 h and go through dehydration, clearing and paraffin embedding. Sections were mounted on positively charged slides after cutting at 4 mm thick, baked at 65 °C for 1h and then stored at room temperature (RT) for later use. Fibrosis was performed using mason’s trichrome (Genmed Scientifics, Wilmington, DE, USA) and Sirius red stain (Servicebio, Wuhan, China) following the protocols of manufactures. For immunofluorescence (IF) assay, the slides were incubated with xylol and descending concentrations of ethanol. Endogenous peroxidases were blocked by using 0.3% H₂O₂ for 10 min at RT. After antigen retrieval, blocking was performed using 5% bovine serum albumin for 30 min at RT. The antibodies used here were listed on the Table S1.

Hydroxyproline assay

The amount of collagen in the lung tissues was determined by a Hydroxyproline Assay Kit according to the manufacturer's protocol (Cat.No.MAK008-1KT, Sigma-Aldrich, St. Louis, MO). Briefly, about 10 mg lung tissues were homogenized in 100 μl water with 100 μl concentrated hydrochloric acid (HCl, 12 M) and hydrolyzed at 120°C for 3 hours. Transfer 20 μl of supernatant at 60°C until completely desiccated. Chloramine T/Oxidation Buffer Mixture was added at room temperature for 5 minutes, followed by the addition of Diluted DMAB Reagent and incubation at 60°C for 90 minutes. Measure the absorbance of samples and standards at 560 nm, and hydroxyproline content was expressed as μg per mg lung tissue.

Statistical analysis

All data were expressed as mean ± SD (standard deviation). For comparisons of different groups, statistical significance was determined by Student's t-test or ANOVA analysis. P-value less than 0.05 was considered statistically significant.

H19 is up-regulated in fibrotic lungs

Analysis of publicly available datasets showed that lncRNA H19 expression was more highly expressed in lung tissue from patients with IPF compared to normal lung tissue, but there was no significant difference between IPF patients and other interstitial lung diseases (ILD) (Figure 1A) [28, 29]. Fibrotic genes, including ACTA2, COL1A2 and MMP7, increasingly expressed in lung tissue from patients with IPF compared to lung tissue from patients with other interstitial lung diseases and to normal lung tissue (Figure 1A) [28, 29]. In addition to, H19 expression increased upon bleomycin-induced lung fibrosis in rats. H19 expression peaked after about 2 weeks of bleomycin-treatment, and then H19 levels returned to an amount comparable to controls (Figure 1B) [30]. In current study, Fluorescent in situ hybridization (FISH) assay showed that H19 expression increased in lungs of bleomycin-treated mice (2 weeks) and located at alveolar epithelium and capillaries (Figure 1C). Furthermore, immunofluorescent staining of surfactant protein C (Sftpc), a marker for type 2 epithelial cells (AEC2s), indicated that H19 was also expressed and up-regulated in AEC2s following bleomycin-treatment (Figure 1C). The qRT-PCR assay confirmed that H19 increasingly expressed in the lungs of mice with bleomycin (BLM) treatment (Figure 1D and 1E).

H19 deficiency represses bleomycin-induced lung inflammatory response

H19 knock out (H19^-/-) mice here were used to elucidate the roles of H19 in bleomycin -induced pulmonary inflammation and fibrosis. After 4-week BLM-treatment, immunofluorescence (IF) staining showed the H19^-/- BLM mice had less CD45⁺ cells accumulated in the lungs than that in lungs of Wt BLM mice (Figure 2A). RT-PCR analysis showed that CD11b and Ccr2 genes expression was reduced significantly in lungs of H19^-/-mice when compared to that of Wt mice (Figure 2B). The inflammatory markers, including F4/80, CD11b, Ccl20, Il1b and Ccr2 increased in Wt BLM mice compared to WT sham mice, but decreased in H19^-/- BLM mice (Figure 2B). Western blot results indicated that protein expression levels of Il6 and p-Stat3 were decreased in lungs of H19^-/- BLM mice relative to the Wt BLM (Figure 2C and 2D).

H19 knockout ameliorates bleomycin-induced pulmonary fibrosis

As shown in Figure 3, histopathological analysis firstly showed reduced fibrosis in the H19^-/- BLM mice (Figure 3A and 3B). The haematoxylin-eosin (H&E) staining and Collagen I immunofluorescence (IF) staining showed the fibrosis increased in Wt BLM mice compared to the Wt sham, but not in lungs of H19^-/- BLM mice (Figure 3A). The Masson’s Trichrome staining and Sirius red staining further indicated that 4-week BLM significantly induced lung fibrosis in WT mice, but had much less impact in H19^-/- mice (Figure 3A). Consistently, quantitation of lung fibrosis in a blinded manner revealed the Ashcroft score decreased significantly in H19^-/- BLM mice when compared to Wt BLM mice (Figure 3B). Furthermore, the pulmonary hydroxyproline levels were significantly increased in Wt BLM mice, but not in H19^-/- BLM mice (Figure 3C). At molecular level, H19^-/-mice had decreased expression of Tgfb1 and Acta2 mRNA in the lungs in relation to Wt animals (Figure 3D). The mRNA levels of Tgfb1, Acta2 and Col1a1 (Figure 3D) and protein expression of Col1a1 (Figure 3E and 3F) reduced in H19^-/- BLM mice compared to the Wt BLM mice.

H19 depletion attenuated the pathways of TGF-β/Smad and S1pr1/Sphk2 in fibrotic lungs

TGF-β/Smad signaling is the key regulating pathway in fibrogenesis [31]. In this study, we showed that the TGF-β mRNA level and protein levels of p-Smad2 and p-Smad3 were increased in the Wt BLM mice, compared with Wt mice, while these proteins reduced in H19^-/- BLM mice (Figure 4A and 4B). Our previous study had reported that S1pr2 and SphK2 played an important role in promoting liver fibrosis [20]. As shown in Figure 4, Western blot results indicated that protein expression levels of S1pr2 and SphK2 were increased in lungs of Wt BLM mice compared to Wt sham mice, but decreased in lungs of H19^-/- BLM (Figure 4C and 4D).

H19 deficiency decreased AEC2s proliferation in lungs of bleomycin-treated mice

In the lung sections from the H19^-/- BLM mice, immunofluorescence results showed that expression of Sftpc was decreased relative to that of the Wt BLM mice (Figure 5A). Additionally, the number of Ki67-positive cells was also reduced in the H19^-/- BLM mice compared to the Wt BLM mice (Figure 5A). Consistently, western blot results indicated that protein expression levels of Sftpc reduced in the H19^-/- BLM mice compared to the Wt BLM mice (Figure 5B and 5C). In addition to, the proteins of p-Egfr decreased in lungs of H19^-/- BLM mice compared to the Wt BLM mice (Figure 5B and 5C).

Presently, the poor understanding in the pathogenesis of IPF has resulted in a lack of effective therapies. In current study, we found that H19 was up-regulated in the fibrotic lungs of IPF patients and bleomycin-treated mice. Functionally, H19 deficiency reduced pulmonary inflammation and fibrosis may via inhibiting Il6/Stat3, TGF-β/Smad or S1pr2/Sphk2. Moreover, H19 deficiency reduced the proliferation of alveolar type II cells in vivo.

H19 is an imprinted and maternally expressed transcript, which is one of the few well-characterized lncRNA [32, 33]. Aberrant expression of H19 has been related to a variety of human diseases [34-38]. The current study showed that IPF patients had higher levels of H19 mRNA in lungs when compared to the control subjects using a public datasets, which indicates that H19 may play an important role in the pathogenesis of IPF. Similarly, H19 mRNA also increased in a mice and rat models of bleomycin-induced pulmonary fibrosis. To elucidate the roles of H19 in the pathogenesis of IPF, we firstly generated a H19 deficiency mouse (H19^-/-). In vivo studies showed that bleomycin-induced pulmonary inflammation was attenuated in H19^-/-mice, demonstrating H19 could promote the inflammatory response after the pulmonary injuries. In this response, CD11b and Ccr2 were reduced in the lung tissues of H19^-/- mice. Li and other colleague recently reported that H19 significantly induced the expression and secretion of chemokine (C-C motif) ligand 2 (CCL-2) that could accumulate the monocytes from circulation into livers [39]. It thus suggests that H19 may promote pulmonary inflammation via attracting the CD11b monocytes into the lung after injures. STAT3 has been reported to play a critical role in the pulmonary inflammation. In the injured lung, STAT3 rapidly activated and increased the production of the proinflammatory molecules IL1β, IL6, TNF-α, iNOS and CCL2 [40-43]. We here showed bleomycin-induced the expression of Il6 and activated Stat3 in the lungs, and these effects were attenuated by H19 depletion. It is indicated H19 deficiency inhibited bleomycin-induced pulmonary inflammation may through attenuating the Il6/Stat3 signaling.

In vivo studies, we further revealed H19 deficiency significantly blocked bleomycin-induced pulmonary fibrosis and reduced the cell proliferation. TGF-β/smad signaling is one of the key pathways responsible for pulmonary fibrosis [31, 44-46]. We here showed that bleomycin induced Tgfb1 mRNA expression and activated the Smad2 and Smad3, but not in the H19^-/- mice. In vitro, a recent study reported that H19 can target miR-140 and regulate the TGF-β/Smad3 pathway [24]. Zhu et. al., reported that H19 could enhance TGF-β signaling in both hepatic stellate cells and hepatocytes and facilitate liver fibrosis [47]. H19 was also showed to accelerate TGF-β1-induced tenogenic differentiation in vitro and promoted tendon healing in a mouse tendon defect model [48]. It thus suggests that H19 enhances the pulmonary fibrosis maybe partly via activation of TGF-β/smad signaling. Sphingosine-1-phosphate and its receptor S1pr2 have been shown to promote lung fibrosis [49-53]. Our previous study showed that H19 could activate the S1pr2/SphK2 signalling pathway in the cholestatic livers. The current study indicated that S1pr2/SphK2 signalling was activated in the bleomycin-treated lungs, but these effects were attenuated by H19 depletion. We suggested that H19 increased lung fibrosis during the bleomycin-treatment partly via downregulation of S1pr2/SphK2 signalling. Epidermal growth factor receptor (EGFR) activating is a major driver of lung adenocarcinoma, which is essential to lung cancer cell proliferation [54]. The present study suggested H19 deficiency represses EGFR activating, which indicating H19 enhances EGFR signaling and lead to promote the cell proliferation in the injured lung.

In summary, we demonstrated that the H19 is a profibrotic lncRNA in the lung and that regulating TGFβ/Smad and S1pr2/Sphk2, two novel mechanisms, underlying H19 activity in pulmonary fibrosis.

IPF, idiopathic pulmonary fibrosis; lncRNA ,long non-coding RNA H19; UIP, usual interstitial pneumonia; RT-PCR, real-time polymerase chain reaction; IF , immunofluorescence; H&E, hematoxylin-eosin; EGFR, Epidermal growth factor ; TGF-β, receptor; transforming growth factor-β; S1PR2, sphingosine-1-phosphate receptor 2 ; SphK2, sphingosine kinase 2; ACTA2, alpha 2 smooth muscle actin; COL1A2, collagen type I alpha 2; MMP7, Matrix metalloproteinase 7

Acknowledgments

The authors extend their gratitude to Shanshan Chen and Yang Liu for their technical support and assistance in performing the experiments.

Author’s contributions

X. Wan and Y. Xiao developed study concept and design, acquisition of data, analysis and interpretation of data. X. Wan, X.Tian, J. Du and Y. Lu performed and analyzed most of the experiments. X. Wan and Y. Xiao wrote the manuscript. All of the authors approved this version of the manuscript to be published.

Funding

This study was supported by the National Natural Science Foundation of China (81770517)

Availability of data and materials

Original data can be requested from corresponding author.

Ethics approval and consent to participate

The animal experiments were approved by the Shanghai Jiao tong University School of Medicine affiliated Xin Hua hospital Animal Care and Use Committee(XHEC-F-2020-008).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interest.

King TE, Jr., Pardo A, Selman M: Idiopathic pulmonary fibrosis. Lancet 2011, 378:1949-1961.
Nalysnyk L, Cid-Ruzafa J, Rotella P, Esser D: Incidence and prevalence of idiopathic pulmonary fibrosis: review of the literature. Eur Respir Rev 2012, 21:355-361.
Fernandez Perez ER, Daniels CE, Schroeder DR, St Sauver J, Hartman TE, Bartholmai BJ, Yi ES, Ryu JH: Incidence, prevalence, and clinical course of idiopathic pulmonary fibrosis: a population-based study. Chest 2010, 137:129-137.
Raghu G, Weycker D, Edelsberg J, Bradford WZ, Oster G: Incidence and prevalence of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2006, 174:810-816.
Travis WD, Costabel U, Hansell DM, King TE, Jr., Lynch DA, Nicholson AG, Ryerson CJ, Ryu JH, Selman M, Wells AU, et al: An official American Thoracic Society/European Respiratory Society statement: Update of the international multidisciplinary classification of the idiopathic interstitial pneumonias. Am J Respir Crit Care Med 2013, 188:733-748.
Ulitsky I, Bartel DP: lincRNAs: genomics, evolution, and mechanisms. Cell 2013, 154:26-46.
Fatica A, Bozzoni I: Long non-coding RNAs: new players in cell differentiation and development. Nat Rev Genet 2014, 15:7-21.
Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L: H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1. Proc Natl Acad Sci U S A 2013, 110:20693-20698.
Ponting CP, Oliver PL, Reik W: Evolution and functions of long noncoding RNAs. Cell 2009, 136:629-641.
Saha P, Verma S, Pathak RU, Mishra RK: Long Noncoding RNAs in Mammalian Development and Diseases. Adv Exp Med Biol 2017, 1008:155-198.
Wu GC, Pan HF, Leng RX, Wang DG, Li XP, Li XM, Ye DQ: Emerging role of long noncoding RNAs in autoimmune diseases. Autoimmun Rev 2015, 14:798-805.
Uchida S, Dimmeler S: Long noncoding RNAs in cardiovascular diseases. Circ Res 2015, 116:737-750.
Pastori C, Wahlestedt C: Involvement of long noncoding RNAs in diseases affecting the central nervous system. RNA Biol 2012, 9:860-870.
Li X, Wu Z, Fu X, Han W: Long Noncoding RNAs: Insights from Biological Features and Functions to Diseases. Med Res Rev 2013, 33:517-553.
Zhang J, Zhu Y, Wang R: Long noncoding RNAs in respiratory diseases. Histol Histopathol 2018, 33:747-756.
Gomez JA, Wapinski OL, Yang YW, Bureau JF, Gopinath S, Monack DM, Chang HY, Brahic M, Kirkegaard K: The NeST long ncRNA controls microbial susceptibility and epigenetic activation of the interferon-gamma locus. Cell 2013, 152:743-754.
Liang WC, Fu WM, Wang YB, Sun YX, Xu LL, Wong CW, Chan KM, Li G, Waye MM, Zhang JF: H19 activates Wnt signaling and promotes osteoblast differentiation by functioning as a competing endogenous RNA. Sci Rep 2016, 6:20121.
Giovarelli M, Bucci G, Ramos A, Bordo D, Wilusz CJ, Chen CY, Puppo M, Briata P, Gherzi R: H19 long noncoding RNA controls the mRNA decay promoting function of KSRP. Proc Natl Acad Sci U S A 2014, 111:E5023-5028.
Li X, Liu R, Yang J, Sun L, Zhang L, Jiang Z, Puri P, Gurley EC, Lai G, Tang Y, et al: The role of long noncoding RNA H19 in gender disparity of cholestatic liver injury in multidrug resistance 2 gene knockout mice. Hepatology 2017, 66:869-884.
Xiao Y, Liu R, Li X, Gurley EC, Hylemon PB, Lu Y, Zhou H, Cai W: Long Noncoding RNA H19 Contributes to Cholangiocyte Proliferation and Cholestatic Liver Fibrosis in Biliary Atresia. Hepatology 2019, 70:1658-1673.
Zhao Y, Feng C, Li Y, Ma Y, Cai R: LncRNA H19 promotes lung cancer proliferation and metastasis by inhibiting miR-200a function. Mol Cell Biochem 2019, 460:1-8.
Huang Z, Lei W, Hu HB, Zhang H, Zhu Y: H19 promotes non-small-cell lung cancer (NSCLC) development through STAT3 signaling via sponging miR-17. J Cell Physiol 2018, 233:6768-6776.
Lu Q, Guo Z, Xie W, Jin W, Zhu D, Chen S, Ren T: The lncRNA H19 Mediates Pulmonary Fibrosis by Regulating the miR-196a/COL1A1 Axis. Inflammation 2018, 41:896-903.
Wang X, Cheng Z, Dai L, Jiang T, Jia L, Jing X, An L, Wang H, Liu M: Knockdown of Long Noncoding RNA H19 Represses the Progress of Pulmonary Fibrosis through the Transforming Growth Factor beta/Smad3 Pathway by Regulating MicroRNA 140. Mol Cell Biol 2019, 39.
Hecker L, Vittal R, Jones T, Jagirdar R, Luckhardt TR, Horowitz JC, Pennathur S, Martinez FJ, Thannickal VJ: NADPH oxidase-4 mediates myofibroblast activation and fibrogenic responses to lung injury. Nat Med 2009, 15:1077-1081.
Koyama K, Goto H, Morizumi S, Kagawa K, Nishimura H, Sato S, Kawano H, Toyoda Y, Ogawa H, Homma S, Nishioka Y: The Tyrosine Kinase Inhibitor TAS-115 Attenuates Bleomycin-induced Lung Fibrosis in Mice. Am J Respir Cell Mol Biol 2019, 60:478-487.
Ashcroft T, Simpson JM, Timbrell V: Simple method of estimating severity of pulmonary fibrosis on a numerical scale. J Clin Pathol 1988, 41:467-470.
Peng X, Moore M, Mathur A, Zhou Y, Sun H, Gan Y, Herazo-Maya JD, Kaminski N, Hu X, Pan H, et al: Plexin C1 deficiency permits synaptotagmin 7-mediated macrophage migration and enhances mammalian lung fibrosis. FASEB J 2016, 30:4056-4070.
Yu G, Tzouvelekis A, Wang R, Herazo-Maya JD, Ibarra GH, Srivastava A, de Castro JPW, DeIuliis G, Ahangari F, Woolard T, et al: Thyroid hormone inhibits lung fibrosis in mice by improving epithelial mitochondrial function. Nat Med 2018, 24:39-49.
Bauer Y, Tedrow J, de Bernard S, Birker-Robaczewska M, Gibson KF, Guardela BJ, Hess P, Klenk A, Lindell KO, Poirey S, et al: A novel genomic signature with translational significance for human idiopathic pulmonary fibrosis. Am J Respir Cell Mol Biol 2015, 52:217-231.
Derynck R, Zhang YE: Smad-dependent and Smad-independent pathways in TGF-beta family signalling. Nature 2003, 425:577-584.
Khosla S, Aitchison A, Gregory R, Allen ND, Feil R: Parental allele-specific chromatin configuration in a boundary-imprinting-control element upstream of the mouse H19 gene. Mol Cell Biol 1999, 19:2556-2566.
Bartolomei MS, Zemel S, Tilghman SM: Parental imprinting of the mouse H19 gene. Nature 1991, 351:153-155.
Stuhlmuller B, Kunisch E, Franz J, Martinez-Gamboa L, Hernandez MM, Pruss A, Ulbrich N, Erdmann VA, Burmester GR, Kinne RW: Detection of oncofetal h19 RNA in rheumatoid arthritis synovial tissue. Am J Pathol 2003, 163:901-911.
Iempridee T: Long non-coding RNA H19 enhances cell proliferation and anchorage-independent growth of cervical cancer cell lines. Exp Biol Med (Maywood) 2017, 242:184-193.
Geng H, Bu HF, Liu F, Wu L, Pfeifer K, Chou PM, Wang X, Sun J, Lu L, Pandey A, et al: In Inflamed Intestinal Tissues and Epithelial Cells, Interleukin 22 Signaling Increases Expression of H19 Long Noncoding RNA, Which Promotes Mucosal Regeneration. Gastroenterology 2018, 155:144-155.
Yoruker EE, Keskin M, Kulle CB, Holdenrieder S, Gezer U: Diagnostic and prognostic value of circulating lncRNA H19 in gastric cancer. Biomed Rep 2018, 9:181-186.
Zhang K, Luo Z, Zhang Y, Zhang L, Wu L, Liu L, Yang J, Song X, Liu J: Circulating lncRNA H19 in plasma as a novel biomarker for breast cancer. Cancer Biomark 2016, 17:187-194.
Li X, Liu R, Wang Y, Zhu W, Zhao D, Wang X, Yang H, Gurley EC, Chen W, Hylemon PB, Zhou H: Cholangiocyte-Derived Exosomal lncRNA H19 Promotes Macrophage Activation and Hepatic Inflammation under Cholestatic Conditions. Cells 2020, 9.
Goodman RB, Pugin J, Lee JS, Matthay MA: Cytokine-mediated inflammation in acute lung injury. Cytokine Growth Factor Rev 2003, 14:523-535.
Severgnini M, Takahashi S, Rozo LM, Homer RJ, Kuhn C, Jhung JW, Perides G, Steer M, Hassoun PM, Fanburg BL, et al: Activation of the STAT pathway in acute lung injury. Am J Physiol Lung Cell Mol Physiol 2004, 286:L1282-1292.
Song Z, Zhao X, Gao Y, Liu M, Hou M, Jin H, Cui Y: Recombinant human brain natriuretic peptide ameliorates trauma-induced acute lung injury via inhibiting JAK/STAT signaling pathway in rats. J Trauma Acute Care Surg 2015, 78:980-987.
Zhao J, Yu H, Liu Y, Gibson SA, Yan Z, Xu X, Gaggar A, Li PK, Li C, Wei S, et al: Protective effect of suppressing STAT3 activity in LPS-induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 2016, 311:L868-L880.
Yao H, Wei S, Xiang Y, Wu Z, Liu W, Wang B, Li X, Xu H, Zhao J, Gao Y: Kangfuxin Oral Liquid Attenuates Bleomycin-Induced Pulmonary Fibrosis via the TGF-beta1/Smad Pathway. Evid Based Complement Alternat Med 2019, 2019:5124026.
Shen ZJ, Braun RK, Hu J, Xie Q, Chu H, Love RB, Stodola LA, Rosenthal LA, Szakaly RJ, Sorkness RL, Malter JS: Pin1 protein regulates Smad protein signaling and pulmonary fibrosis. J Biol Chem 2012, 287:23294-23305.
Higashiyama H, Yoshimoto D, Okamoto Y, Kikkawa H, Asano S, Kinoshita M: Receptor-activated Smad localisation in bleomycin-induced pulmonary fibrosis. J Clin Pathol 2007, 60:283-289.
Zhu J, Luo Z, Pan Y, Zheng W, Li W, Zhang Z, Xiong P, Xu D, Du M, Wang B, et al: H19/miR-148a/USP4 axis facilitates liver fibrosis by enhancing TGF-beta signaling in both hepatic stellate cells and hepatocytes. J Cell Physiol 2019, 234:9698-9710.
Lu YF, Liu Y, Fu WM, Xu J, Wang B, Sun YX, Wu TY, Xu LL, Chan KM, Zhang JF, Li G: Long noncoding RNA H19 accelerates tenogenic differentiation and promotes tendon healing through targeting miR-29b-3p and activating TGF-beta1 signaling. FASEB J 2017, 31:954-964.
Huang LS, Sudhadevi T, Fu P, Punathil-Kannan PK, Ebenezer DL, Ramchandran R, Putherickal V, Cheresh P, Zhou G, Ha AW, et al: Sphingosine Kinase 1/S1P Signaling Contributes to Pulmonary Fibrosis by Activating Hippo/YAP Pathway and Mitochondrial Reactive Oxygen Species in Lung Fibroblasts. Int J Mol Sci 2020, 21.
Park SJ, Im DS: Deficiency of Sphingosine-1-Phosphate Receptor 2 (S1P2) Attenuates Bleomycin-Induced Pulmonary Fibrosis. Biomol Ther (Seoul) 2019, 27:318-326.
Zhao J, Okamoto Y, Asano Y, Ishimaru K, Aki S, Yoshioka K, Takuwa N, Wada T, Inagaki Y, Takahashi C, et al: Sphingosine-1-phosphate receptor-2 facilitates pulmonary fibrosis through potentiating IL-13 pathway in macrophages. PLoS One 2018, 13:e0197604.
Huang LS, Berdyshev EV, Tran JT, Xie L, Chen J, Ebenezer DL, Mathew B, Gorshkova I, Zhang W, Reddy SP, et al: Sphingosine-1-phosphate lyase is an endogenous suppressor of pulmonary fibrosis: role of S1P signalling and autophagy. Thorax 2015, 70:1138-1148.
Milara J, Navarro R, Juan G, Peiro T, Serrano A, Ramon M, Morcillo E, Cortijo J: Sphingosine-1-phosphate is increased in patients with idiopathic pulmonary fibrosis and mediates epithelial to mesenchymal transition. Thorax 2012, 67:147-156.
Stella GM, Luisetti M, Inghilleri S, Cemmi F, Scabini R, Zorzetto M, Pozzi E: Targeting EGFR in non-small-cell lung cancer: lessons, experiences, strategies. Respir Med 2012, 106:173-183.

Supplements.docx

Download PDF

Journal Publication

published 02 Nov, 2020

Read the published version in Respiratory Research →

Reviewer #2 agreed at journal
30 Sep, 2020
Review #2 received at journal
30 Sep, 2020
Editorial decision: Minor revision
30 Sep, 2020
Reviewers invited by journal
29 Sep, 2020
Reviewer #1 agreed at journal
29 Sep, 2020
Review #1 received at journal
29 Sep, 2020
Editor assigned by journal
27 Sep, 2020
Submission checks completed at journal
26 Sep, 2020
Editor invited by journal
26 Sep, 2020

You are reading this older preprint version

Read the latest preprint version →

Long non-coding RNA H19 deficiency ameliorates bleomycin-induced pulmonary inflammation and fibrosis

Status:

Journal Publication

Version 4

Abstract

Figures

Background

Materials And Methods

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

Status:

Journal Publication

Version 4