Silencing of long noncoding RNA H19 alleviates pulmonary injury, inflammation and fibrosis in acute respiratory distress syndrome rats through regulating MicroRNA-423-5p

Background: This study aimed to explore the function of long noncoding RNA H19 (H19) on pulmonary injury, inflammation and fibrosis in lipoproteins (LPS)-induced acute respiratory distress syndrome (ARDS) rats. Methods: The LPS-induced ARDS rat model was established by intratracheal instillation with 2 mg/kg LPS. QRT-PCR was performed to detect the expression of H19, miR-423-5p, tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6,, monocyte chemoattractant protein (MCP)-1 and vascular endothelial growth factor (VEGF). Histology score was detected by hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of proinflammatory cytokines and the concentration of VEGF in bronchoalveolar lavage fluid (BALF). The protein expression of fiber factors was measured by western blot. The degree of fibrosis was observed by masson-trichrome staining. Dual-luciferase reporter assay was used to determine the binding site between miR-423-5p and H19. Results: The expression of H19 was significantly increased, while miR-423-5p was decreased in LPS-induced ARDS rats. Silencing of H19 decreased the mRNA expression of TNF-α, IL-1β, IL-6, MCP-1 and VEGF in LPS-induced ARDS rats, and decreased the levels of TNF-α, IL-1β, IL-6and the concentration of VEGF in BALF, histology score of LPS-induced ARDS rats. H19 inhibition also decreased the fibrosis score and the proteins expression of fiber factors of LPS-induced ARDS rats. Furthermore, miR-423-5p eliminated the effect of H19 on LPS-induced MH-S cells. Conclusions: Silencing of H19 ameliorated the pulmonary injury, inflammation and fibrosis of LPS-induced ARDS through regulating miR-423-5p,

3 Background Acute respiratory distress syndrome (ARDS) is characterized by severe hypoxaemia, pulmonary inflammation, alveolar edema and pulmonary fibrosis [1]. ARDS has a mortality of 20%~50% with poor prognosis worldwide [2]. ARDS patients require mechanical ventilatory support that carries a high financial cost [3]. There are extremely few effective treatments for ARDS mainly due to its complex pathogenesis and timely diagnosis except lung preservation strategies [4,5]. Herein, it is necessary to explore new strategies or promising biomarkers for ARDS treatment and diagnosis.
Long noncoding RNAs (lncRNAs) are a class of noncoding RNA transcripts with a minimum length of 200 nucleotides and limited protein-coding capacity [6,7]. Increasing researches demonstrate that lncRNAs could take part in the pathogenesis of many pulmonary diseases, such as lncRNA MALAT1 in acute lung injury (ALI) [8], lncRNAPCAT29 in pulmonary fibrosis [9] and lncRNA TUG1 in chronic obstructive pulmonary disease [10].
LncRNA H19 (H19) as one of the lncRNAs has been found to be involved in several pulmonary diseases. For instance, knockdown of H19 alleviates pulmonary fibrosis through regulating the miR-196a/COL1A1 axis [11]. Zhang et al. suggested that H19 can promote epithelial mesenchymal transition (EMT) in lung cancer through targeting miR-484 [12].
However, the underlying mechanism and biological influence of H19 in the regulation of ARDS progression remain still limited.
LncRNAs modulate the expression of mRNAs via regulating its translation and degradation through interacting with microRNAs (miRNAs) [13]. MiRNAs are series of small, noncoding RNAs with a length of 1825 nt [14]. Numerous studies have proved the key function of miRNAs in the progresses of ALI. MiR-27a alleviates the inflammatory responses of pulmonary in LPS-induced ALI via regulating TLR4/MyD88/NF-κB pathway [15].
Overexpression of miR-125b ameliorates the inflammation and histopathology changes of 4 pulmonary in LPS-induced ALI [16]. MiR-144-3p protects mice from LPS-induced ALI via reducing lung epithelial cell apoptosis [17]. ARDS is a severe clinical syndrome of ALI, and affects both surgical and medical patients [18]. However, the mediatory relationship between H19 and miR-423-5p on ARDS remains unclear.
Here, we determined the function of H19 on the pulmonary injury, inflammation and fibrosis in lipoproteins (LPS)-induced ARDS rats. We found that miR-423-5p is a target gene of H19. Then we explored the regulatory effect of miR-423-5p on the expression of proinflammatory cytokines, CAMs, chemotactic factors, VEGF and the proteins expression of fiber factors in LPS-induced MH-S cells. Our research may discover a hopeful therapeutic target for ARDS, and broaden our horizons of the underlying mechanisms responsible for ARDS treatment.

Animals
Twenty male wistar rats (180 ± 20 g) were purchased from Institute of Zoology, Chinese Academy of Sciences (Beijing, China). Rats were fed standard chow and water, and maintained under the light/dark cycle (12 h/12 h). This study was performed with the approval of our hospital Animal Ethics Committee.

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Sham group rats (n = 5) were intratracheally instilled with an equal volume of normal saline (NS). Blood samples, lung tissues and bronchoalveolar lavage fluid (BALF) were collected for the future experiments.

Histopathology detection by hematoxylin-eosin (HE) staining
Lung samples were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin, cut into sections at 6 µm thickness, and stained with HE staining. By means of light microscopy, the histopathological change of lung tissues was observed. The histology score of lung tissues was evaluated as previously described [19].
Analysis of arterial blood gas and ratio of lung wet/dry (W/D) weight Samples of arterial blood were obtained from carotid artery. Partial pressure of arterial oxygen (PaO 2 ) and partial pressure of arterial carbon dioxide (PaCO 2 ) were detected by automatic blood gas analyzer (Radiometer, Copenhagen, Denmark). Lungs were weighed and subsequently dried in a 60 °C oven for 72 hours. The ratio of lung W/D weight  Table 1. Table 1 Primer sequences used in quantitative real-time PCR Name of primer

Dual-luciferase reporter assay
The potential binding sites of miR-423-5p and H19 were predicted according to

Statistical analysis
Statistical analysis was performed with SPSS 23.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism software 7.0 (San Diego, CA, USA). Data was presented as mean ± SD. The differences between various groups were analyzed by one-way ANOVA followed by the multiple comparisons test. The data of two groups were assessed using Student's t-test. A P value < 0.05 was considered statistically significant.

Silencing of H19 alleviates pulmonary injury of LPS-induced ARDS
LPS-induced ARDS model in rat was established by induction of LPS. QRT-PCR showed that the expression of H19 was markedly up-regulated in the ARDS group (P < 0.01) (Fig. 1A).
As shown in Fig. 1B, the expression of H19 was markedly down-regulated in the ARDS + sh-H19 group compared with the ARDS group (P < 0.001), suggesting that the transfection was succeed, and the transfection of sh-H19 markedly down-regulated the relative expression of H19. The histopathological change and histology score of lung tissues were observed by HE staining. Compared with the sham group, the inflammatory cells 9 infiltration and interstitial edema were obviously in the ARDS group. Treatment with sh-H19 markedly alleviated the inflammatory cells infiltration and interstitial edema ( Fig. 1C). By contrast to the sham group, the histology score was markedly elevated in the ARDS group (P < 0.001) (Fig. 1D). Silencing of H19 markedly reduced the histology score of lung tissues (P < 0.01) (Fig. 1D). Additionally, the level of blood PaO 2 was markedly reduced and PaCO 2 , ratio of lung W/D weight and lung edema score were increased in the ARDS group comparing with the sham group (P < 0.001) (Fig. 1E-H). H19 inhibition markedly elevated PaO 2 level and decreased PaCO 2 , ratio of lung W/D weight and lung edema score (P < 0.05 or P < 0.01) ( Fig. 1E-H).

H19 inhibition attenuates pulmonary inflammation of LPS-induced ARDS rats
To evaluate the effect of H19 inhibition on pulmonary inflammation of LPS-induced ARDS rats, we examined the content of TP, TNF-α, IL-6 and IL-1βby ELISA. Compared with the sham group, the levels of TP, TNF-α, IL-6 and IL-1β in BALF were significantly increased in the ARDS group (P < 0.001). The above indexes in BALF in the ARDS + sh-H19 group were markedly lower than that in the ARDS group (P < 0.001) ( Fig. 2A-D). The mRNA expression levels of TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1 and MCP-1 in lung tissues was measured by qRT-PCR. The mRNA expression levels of TNF-α, IL-6, IL-1β, ICAM-1, VCAM-1 and MCP-1 in lung tissues was significantly up-regulated in the ARDS group by contrast to the sham group. Silencing of H19 markedly down-regulated the above indexes in lung tissues (P < 0.001) (Fig. 2E-J). Immunohistochemical assay showed that staining intensity of VEGF was markedly increased in the ARDS group compared with the sham group. The staining intensity of VEGF in the ARDS + sh-H19 group was markedly lower than that in the ARDS group (P < 0.001) (Fig. 2K and L). The concentration of VEGF in BALF and the mRNA expression level of VEGF in lung tissues were detected by ELISA and qRT-PCR, respectively. The concentration of VEGF in BALF and the mRNA expression level of VEGF in lung tissues was markedly up-regulated in the ARDS group compared with the sham group (P < 0.001). Knockdown of H19 markedly reduced the concentration of VEGF in BALF and mRNA expression level of VEGF in lung tissues (P < 0.01) (Fig. 2M and N). MiR-423-5p is a direct target of H19 StarBase3.0 was utilized to predict the relationship between miR-423-5p and H19, and the sequence of binding site was showed in Fig. 4A. Dual-luciferase reporter assay suggested the luciferase activity of MH-S and MLE-12 cells co-transfected with miR-423-5p mimics and H19 Wt was significantly decreased (P < 0.001) while there was none difference after miR-423-5p mimics and H19 Mut co-transfection (P > 0.05) ( Fig. 4B and C). The expression of miR-423-5p in lung tissues, lung cells (MH-S and MLE-12 cells) and H19 in lung cells were measured by qRT-PCR. The expression of miR-423-5p in lung tissues was significantly down-regulated in the ARDS group. Silencing of H19 markedly promoted the expression of miR-423-5p in lung tissues (P < 0.001) (Fig. 4D). Compared with mock group, the expression of H19 in MH-S and MLE-12 cells was significantly down-regulated in the si-H19 group (P < 0.001) (Fig. 4E and F). Knockdown of H19 increased the expression of miR-423-5p in MH-S and MLE-12 cells (P < 0.001) (Fig. 4G and H).

MiR-423-5p eliminates the effect of H19 on the LPS-stimulated MH-S cells
In order to explore the potential mechanism of H19/miR-423-5p axis, we firstly performed qRT-PCR analysis for H19 and miR-423-5p levels in MH-S cells. By contrast to the control group, the expression of H19 in MH-S cells was markedly up-regulated in the LPS group (P < 0.001) (Fig. 5A), and miR-423-5p was down-regulated (P < 0.001) (Fig. 5B). The expression of H19 in MH-S cells in the LPS + si-H19 group was lower than that in the LPS group (P < 0.001) (Fig. 5C). The transfection of miR-423-5p inhibitor inhibited the expression of miR-423-5p in MH-S cells (P < 0.001) (Fig. 5D). After detection of inflammatory factor and pulmonary fibrosis associated protein, we found that the mRNA    VEGF in lung tissues was measured by qRT-PCR. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Sham; ## P < 0.01, ### P < 0.001 vs. ARDS.