The effect of H19-miR-29b interaction on bleomycin-induced mouse model of idiopathic pulmonary fibrosis

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Abstract

Pulmonary fibrosis, characterized by the destruction of lung tissue architecture and the formation of fibrous foci, currently has no satisfactory treatment. Emodin is a component of Chinese herb that has been reported to be medicable on pancreatic fibrosis and liver fibrosis. However, its role in pulmonary fibrosis has not been reported yet. In the present study, we investigated the hypothesis that H19 play a promotive role in bleomycin-induced epithelial-mesenchymal transition of alveolar epithelial cell, and H19 exerts its effect through miR-29b regulation. H19 expression was positively correlated with COL1A1 and Acta2 expression; H19 knockdown inhibited COL1A1 and Acta2 expression. Moreover, H19 interacted with miR-29b through directly binding to the 3′UTR; miR-29b inhibited COL1A1 expression by directly binding to the 3′UTR. In conclusion, we revealed the promotive effect of H19 on BLM-induced IPF, and demonstrated the mechanism by which H19/miR-29b interaction exerts its effect on regulating pulmonary fibrosis. The present study provided a potential therapy to treat IPF.

Introduction

Idiopathic pulmonary fibrosis (IPF), a progressive and diffuse parenchymal disease with a high mortality rate [1], [2], is a fatal disease with no known cause and no effective pharmacological treatment. Bleomycin (BLM) has been used to induce lung fibrosis, which mimics IPF, in both mouse and rat models [3], [4]. Pulmonary fibrosis is a common characteristic of IPF and systemic sclerosis with interstitial lung disease (SSc-ILD) [5]. Pulmonary fibrosis is an incurable and irreversible lung disease [6], and new insights into the molecular mechanisms underlying the development of pulmonary fibrosis are needed to develop both novel therapeutic strategies and early diagnostic and prognostic biomarkers.

Human genome sequence data indicates that more than 90% of the DNA sequences actively transcribed but only 2% of it encodes protein, thus the majority of transcripts are referred to as non-coding RNAs (ncRNAs) [7], [8]. Recent studies have shown that long non-coding RNAs (lncRNAs) play important roles in both normal development and diseases including cancer [9]. LncRNAs have emerged as new players in cancer research and several studies have shown that some lncRNAs function as oncogenes, tumor suppressor genes or both, depending on the circumstance [10]. Among the large amount of lncRNAs, H19 has been reported to be closely related with diverse diseases, including fibrosis [11], [12]. Xie et al. revealed that H19 up-regulation contributes to renal fibrosis. H19 inhibition might represent a novel anti-fibrotic treatment in renal diseases [12]. However, there are few reports about the research of the effect of H19 on pulmonary fibrosis.

The mechanisms by which lncRNAs exert their effect varies under different conditions, however, emerging evidences have revealed that the interaction between lncRNAs and microRNAs plays a major role [13], [14]. Zhu et al. reported that lncRNA H19/miR-675 axis represses prostate cancer metastasis by targeting TGFBI [15]. The interaction between miR-141 and lncRNA-H19 has been regarded as an important component in regulating cell proliferation and migration in gastric cancer [16].

MiRNAs play an important role in lung cells, where they have been implicated in cell proliferation, apoptosis, differentiation, etiology and the progression of fibrotic diseases [17]. H19 has been predicted to bind with miR-29b by miRcode. miR-29b has been regarded as a key regulator of fibrosis. Zhang et al. reported that loss of miR-29 is associated with cardiac fibrosis, and that miR-29b plays a protective role in AngII-mediated cardiac remodeling and may be a therapeutic agent for cardiac fibrosis by targeting the TGF-β/Smad3 pathway [18]. Moreover, Cushing et al. suggests a role of miR-29 in the pathogenesis of pulmonary fibrosis. miR-29 may be a potential new therapeutic target for this disease [19].

In the present study, we hypothesized that the interaction of H19 and miR-29b may be included in lung fibrosis. We demonstrated that the interaction of H19 and miR-29b is involved in pulmonary fibrosis in a bleomycin-induced mouse model; and identified the potential molecular mechanisms by which H19/miR-29b regulates pulmonary fibrosis. We found that treatment of NIH3T3 cells with miR-29b inhibited IPF related factor expression and attenuated the effects of H19.

Section snippets

Cell culture

Mouse fibroblast cells (NIH3T3) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). The cells were frozen at an early passage and cultured for a maximum of eight passages. The cells were maintained at 37 °C with 5% v/v CO2 in Dulbecco's modified Eagle's medium (DMEM, Life Technologies/Gibco, Grand Island, NY) supplemented with 10% fetal calf serum (FCS, Life Technologies/Gibco, Grand Island, NY), 100 U/ml penicillin G and 100 μg/ml streptomycin (Life

Results

Knockdown of H19 inhibited IPF progress and the expression of IPF related factors in BLM-induced mouse model of IPF.

To investigate the effect of H19 in IPF, Bleomycin-induced mouse model of lung fibrosis was established as verified by H&E-staining and Masson's trichrome assays for collagen deposition (Fig. 1A). The expression levels of H19, COL1A1 and Acta2 were up-regulated in BLM-induced mouse model of IPF, compared with normal control (Fig. 1B). Moreover, a positive correlation between the

Discussion

Recently, more and more studies demonstrated that IPF could be regulated by lncRNAs [20]. Song et al. reported that the expression levels of N4bp2 and Plxna4 significantly increased in fibrotic rats, and were highly correlated with those of MRAK088388 and MRAK081523, respectively. Among their shared miRNAs, miR-29b-3p and let-7i-5p decreased in the model group, and were negatively correlated with the expression of MRAK088388 and MRAK081523, respectively [20]. In this study, we found that the

Acknowledgement

This investigation was supported by National Key Scientific & Technology Support Program:Collaborative innovation of Clinical Research for chronic obstructive pulmonary disease and lung cancer (No. 2013BAI09B09). This investigation was supported by the program of Hunan Provincial Department of Science and Technology Support (project number: 2012FJ4366; 2011SK3224).

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