The upregulation of miR-204-3p in LPS-induced acute lung injury aggravated pulmonary endothelial cells apoptosis via targeting sulfatase 2

1Department of Emergency Medicine, Nanjing Medical University Affiliated Changzhou No. 2 People’s Hospital, Changzhou, Jiangsu Province, 213164, China; 2Department of Pediatrics, Nanjing Medical University Affiliated Changzhou No. 2 People’s Hospital, Changzhou, Jiangsu Province, 213164, China; 3Department of Respiratory Medicine, Nanjing Medical University Affiliated Changzhou No. 2 People’s Hospital, Changzhou, Jiangsu Province, 213164, China


INTRODUCTION
Acute lung injury (ALI) is the damage of alveolar epithelial cells and pulmonary capillary endothelial cells caused by non-cardiogenic factors. ALI is characterized by alveolar-capillary barrier damage, bilateral pulmonary infiltrates, non-cardiogenic pulmonary edema, poor lung compliance, and imbalance of ventilation/blood flow ratio (Ferguson et al., 2005;Levitt & Matthay, 2006;Albus, 2012;Brown & Watson, 2018). Patients with ALI often suffer from respiratory distress and refractory hypoxemia. There are various factors causing ALI, such as sepsis, pneumonia, aspiration of gastric contents, severe trauma, acute pancreatitis, and blood transfusion (Brown & Watson, 2018). Indirect ALI is a common consequence of sepsis and about 40% of sepsis patients suffer from ALI (Brown & Watson, 2018;Wang et al., 2019). Sepsis is caused by Gram-negative bacteria and lipopolysaccharide (LPS) plays a key role in the inflammatory response of ALI (Brown & Watson, 2018;Wang et al., 2019). Therefore, LPS can be used to establish the mouse ALI model. ALI may further develop into the severe stage named acute respiratory distress syndrome (ARDS) if not wellcontrolled (Ferguson et al., 2005;Levitt & Matthay, 2006;Albus, 2012;Brown & Watson, 2018). Although recently progress has been made in the diagnosis and treatment of ALI/ARDS, the mortality rate of ALI is still high, ranging from 29% to 42% depending on the therapeutic strategy and age (Ferguson et al., 2005;Levitt & Matthay, 2006;Albus, 2012;Brown & Watson, 2018). Therefore, it is urgent to find a more effective drug to control the development of ALI.
MicroRNAs (miRNAs) belong to non-coding RNAs (ncRNAs) that do not encode proteins and have a length of about 22 nt (Sotiropoulou et al., 2009;Zhao & Li, 2019). MiRNAs play an important role in the expression of 30% of protein-coding genes in vivo through interacting with the mRNA of the target gene at the 3′-untranslated region to initiate the inhibition of mRNA translation or induction of mRNA degradation (Sochor et al., 2014;). An increasing number of studies have reported the role of miRNAs in the regulation of ALI (Hara et al., 2007). For example, the inhibition of miRNA-494 alleviated ALI (Ling et al., 2018); upregulation of miR-146a suppressed the inflammatory responses in LPS-induced ALI (Zeng et al., 2013); miR-132 inhibited LPS-induced inflammation in alveolar macrophages (Liu et al., 2015); the inhibition of miR-34b-5p attenuated lung inflammation in ALI mouse model induced by LPS (Xie et al., 2018); downregulation of miR-181a attenuated LPS-induced ALI by targeting Bcl-2 . The accumulating evidence revealed that miRNAs exert an important role in the regulation of ALI.
In this study, we found that ALI could be alleviated by down-regulating miR-204-3p and aggravated by overexpressing miR-204-3p. Therefore, the inhibition of miR-204-3p may be a therapeutic strategy for ALI.

LPS-induced ALI mouse model
Male C57BL6/J mice (8-10 weeks old) (n=5 per group) were anaesthetized and intratracheally instilled with 10 mg/kg LPS. Control mice were injected with equivalent pyrogen-free PBS. Saline was injected into the lung and immediately suctioned out to collect the alveolar lavage fluid. All animal experiments were approved by the Ethics Committee of Nanjing Medical University Affiliated Changzhou No.2 People's Hospital for the use of animals and conducted in accordance with the National Institutes of Health Laboratory Animal Care and Use Guidelines.

Enzyme-linked immunosorbent assay (ELISA)
The levels of TNF-α, IL-6 and IL-1β in alveolar lavage fluid were assessed with ELISA kits purchased from Thermo Fisher Scientific. Each sample was measured in three technical replicates.

Western blotting
The cell lysate was collected by centrifugation at 12 000×g for 10 min after cells were lysed in radio-immunoprecipitation assay buffer (RIPA buffer) supplemented with protease inhibitor cocktail on ice for 30 min. Bicinchoninic acid assay (BCA assay) was used to quantify the protein concentration in the supernatant. Proteins were separated by SDS-PAGE, then transferred onto PVDF membranes. After blocking with 5% (w/v) dry non-fat milk and the incubation with primary antibodies, secondary antibodies were applied to the membranes. Blots were visualized with enhanced chemiluminescence. Relative protein expression was quantified with ImageJ software. β-actin served as the internal control. Primary antibodies against β-actin (ab8226), Bax (ab32503), Bcl-2 (ab32124), cleaved caspase-3 (ab2302), SULF2 (ab113405) and secondary antibody conjugated to horseradish peroxidase (HRP) were supplied by Abcam (China).

Luciferase assay
Cells were collected, rinsed twice with PBS, resuspended in Reporter Lysis Buffer (Promega Corporation, Madison, USA) at 1×10 6 /100 µl, and incubated on ice for 20 min. The supernatant was collected after the centrifugation at 12 000×g for 20 min and mixed with Luciferase Assay Reagent at the ratio of 1:5. The luminescence was detected with a luminometer.

Hematoxylin-eosin (HE) staining
Lung tissue was fixed for 24 h and dehydrated with xylene. The dehydrated tissue was embedded in paraffin, sliced, dried, and dewaxed. HE staining was performed using hematoxylin for 5 min. After washing with distilled water, the color of hematoxylin was separated by alcohol hydrochloric acid (0.5-0.1% hydrochloric acid diluted with 70% alcohol). Then the sections were stained with eosin for 2 min, followed by dehydration, clearing, drying and mounting. An optical microscope was used to photograph the histological changes.

Cell viability
A549 cells were transfected with miR-204-3p mimic or miR-204-3p inhibitor, then seeded at a concentration of 8×10 4 cells/ml into a 96-well plate (100 μl per well). After incubation for 24 h, cells were treated with 1 μg/ml of LPS for another 24 h. CCK-8 (Abcam, China) solution was added to the 96-well plate (10 μl per well). The plate was incubated at 37°C for 1-4 hours. A 450 nm was measured using a microplate reader.

Statistical analysis
Data are presented as mean ± S.E.M. Statistical significance was analyzed using Student's t-test. p<0.05 was considered to be statistically significant.

miR-204-3p was up-regulated in the ALI mouse model induced by LPS
ALI mouse model was established by LPS intratracheal injection. ELISA assay revealed that secretion of TNF-α, IL-6 and IL-1β cytokines increased in the ALI mouse model (Fig. 1A). The protein expression of Bax and cleaved caspase 3 enhanced while the expression of Bcl-2 decreased after LPS treatment (Fig. 1B). HE staining demonstrated that normal lung tissue structure was clear and the alveolar space was large, but after LPS treatment, the lung tissue structure was abnormal and the alveolar structure was unobvious (Fig. 1C). The relative expression of miR-204-3p was increased by LPS treatment (Fig. 1D). These results suggested that LPS was able to induce lung injury and increase the expression level of miR-204-3p.

miR-204-3p regulated LPS-induced cell apoptosis via SULF2
To verify the interaction of miR-204-3p and SULF2, and its effect on LPS-induced cell death, A549 cells were used for the corresponding tests. In LPS-treated A549 cells, the secretion of TNF-α, IL-6 and IL-1β cytokine was increased, and the effect was reduced by anti-miR-204-3p treatment and promoted by miR-204-3p mimic (Fig. 4A). LPS treatment induced cell death, which was prevented by the inhibitor of miR-204-3p and aggravated by miR-204-3p overexpression (Fig. 4B). In LPS-treated A549 cells, the treatment with miR-204-3p inhibitor elevated the expression of SULF2 and Bcl-2, which was prevented by miR-204-3p overexpression (Fig. 4C). These results suggested that miR-204-3p regulated LPSinduced cell apoptosis via SULF2.

DISCUSSION
ALI is the damage of alveolar epithelial cells and pulmonary capillary endothelial cells (Ferguson et al., 2005;Levitt & Matthay, 2006;Albus, 2012;Brown & Watson, 2018). It can be caused by various factors, such as sepsis, pneumonia, aspiration of stomach contents, acute pancreatitis, and blood transfusion (Brown & Watson 2018). ALI can progress into ARDS which is characterized by a high mortality rate (Ferguson et al., 2005;Levitt & Matthay, 2006;Albus, 2012;Brown & Watson, 2018). Increasing evidence demonstrated that microRNAs control the expression of many protein-coding genes and play an important role in the regulation of various diseases including ALI (Hara et al., 2007;Sochor et al., 2014;. By removing 6-O-sulfates from specific heparan sulfate intrachain sites, SULF2 regulates the functions of many growth factors and cytokines (Yue, 2017). SULF2 was proved to be involved in many signaling pathways by mobilizing protein ligands from the sequestration by heparan sulfate proteoglycans (HSPGs) thus liberating the ligands for binding to signal transduction receptors, and SULF2 was also identified as a driver of carcinogenesis (Lui et al., 2016;Li et al., 2019). SULF2 was overexpressed in the idiopathic pulmonary fibrosis lungs.
Knockdown of SULF2 aggravated lung injury induced by bleomycin, demonstrating that SULF2 protected hyperplastic type II alveolar epithelial cells from epithelial injury, inflammation, and cell death (Yue, 2017). However, the protective mechanism of SULF2 on pulmonary endothelial cells remains to be further investigated. We found that the expression of miR-204-3p was upregulated in LPS-induced ALI and miR-204-3p targeted and inhibited the expression of SULF2. MicroRNAs inhibit cancer development by targeting SULF2 (Ling et al., 2018;Li et al., 2019). In this work, the role of micro-RNA in the regulation of ALI and the possible involvement of SULF2 were investigated.
LPS was used to induce an inflammatory response to establish the ALI mouse model. In this study, the expression of miR-204-3p was upregulated in LPS-induced ALI mouse model, indicating that the expression level of miR-204-3p may be used as a biomarker for ALI diagnosis. Correspondingly, the secretion of TNF-α, IL-6 and IL-1β cytokines was increased in the ALI mice model, suggesting that the inflammatory response was induced by LPS treatment. The protein expression level of Bax and cleaved caspase 3 increased while the expression of Bcl-2 decreased after LPS treatment, suggesting that LPS treatment induced cell apoptosis. To further investigate the role of miR-204-3p in ALI, the expression of miR-204-3p was downregulated or upregulated in vivo and in vitro. The cytokine secretion was inhibited by downregulation of miR-204-3p and promoted by miR-204-3p overexpression. Moreover, lung cell apoptosis was prevented by miR-204-3p inhibition and promoted by miR-204-3p overexpression. The lung tissue in LPS-treated mice exhibited abnormal structure and unobvious alveolar structure, which was alleviated by downregulation of miR-204-3p and deteriorated by miR-204-3p overexpression. These results demonstrated that miR-204-3p aggravated the development of ALI and targeting miR-204-3p may be a therapeutic strategy for ALI treatment. The potential regulatory mechanism of miR-204-3p in ALI was further explored. Luciferase assay suggested that miR-204-3p targeted and negatively regulated the expression of SULF2, indicating that overexpression of miR-204-3p promoted ALI via inactivation of the expression of SULF2.
In the present study, we verified that miR-204-3p was upregulated in ALI and miR-204-3p could target and inhibit the expression of SULF2. This finding provided a molecular target for the treatment of ALI.