MiR-490 alleviates sepsis-induced acute lung injury by targeting MRP4 in new-born mice

The aim of this study was to investigate whether the effects of miR-490 on acute lung injury (ALI) induced by sepsis in vitro and in vivo were through targeting multi-drug resistance-associated protein 4 (MRP4). MiR-490 agomir/NC agomir was injected into mice before cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with or without miR-490 agomir/NC agomir/ MRP4/empty vector before lipopolysaccharide (LPS) stimulation. Histopathology, injure score, and Wet/ Dry (W/D) of lung tissues were assessed. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β level in bronchoalveolar lavage fluid (BALF) were measured. The levels of caspase-3, Bcl-2, TNF-α, and IL-1β were measured in MPVECs. Dual-luciferase reporter assay was used to analyze the relationship between MRP4 and miR-490. When compared to the sham group, in CLP mice, the alveolar lung tissue showed significantly hyperemic, alveolar collapse, the W/D ratio was increased, and the injury index was increased. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β levels were significantly increased in BALF from CLP mice. The levels of TNF-α and IL-1β were significantly increased in lung tissue from CLP mice. Overexpression of miR-490 alleviated lung injury caused by CLP and inhibited inflammation in mice. The levels of TNF-α, IL-1β and caspase-3 were significantly increased, but the level of Bcl-2 was significantly decreased in MPVECs treated with LPS compared to the control group. Overexpression of miR-490 also reversed the increase of TNF-α, IL-1β, cleaved caspase-3 and Bcl-2 caused by LPS in MPVECs. Dual-luciferase reporter assay confirmed that the target gene of miR-490 was MRP4. Besides, overexpression of MRP4 upregulated TNF-α, IL-1β, and cleaved caspase-3, but downregulated the increase of Bcl-2 induced by miR-490 agomir transfection. These data suggested that miR-490 could relieve sepsis-induced acute lung injury in neonatal mice via targeting MRP4.


INTRODUCTION
Sepsis, a life-threatening disease, is characterized by multiple organ failure and is a common condition in the intensive care units (ICU), especially in pediatric intensive care units (PICU) (Zhang et al., 2012;Sagy et al., 2013;Shin et al., 2018). There are more than 18 million cases of severe sepsis worldwide annually and the mortality rate is still as high as 30-70% (Aziz et al., 2018). Therefore, it is essential to better understand the pathogenesis and develop new treatments for sepsis.
The lung is the most vulnerable target organ in patients with sepsis, and acute lung injury (ALI)/acute respiratory distress syndrome (ARDS) can occur early in patients with sepsis (Ning et al., 2017). ALI is characterized by an over-activated inflammatory reaction in the lungs that leads to severe damage to the body's gas exchange function, damaged alveolar-capillary vascular barriers, and pulmonary edema (Amin & Rahmawati 2017). Pulmonary microvascular endothelial cells (PMVECs) are the target and effector cells of the lung injury process. Furthermore, mice model of sepsis caused by cecal ligation and puncture (CLP) and ALI cellular model induced by lipopolysaccharide (LPS) have been widely and successfully used (Meng et al., 2019). During sepsis-induced ALI, some cytokines and mediators of inflammation can destroy the intercellular junctions, alter the cytoskeletal structure or destroy the cell monolayer, ultimately causing the gap in the endothelium, microvascular leakage and lung damage (Zhang et al., 2012).
MicroRNAs (miRNAs) are a class of evolutionary highly conserved non-coding RNA molecules, which play vital roles in various physiological processes, including organ syndrome induced by sepsis, especially in ALI (Kingsley & Bhat 2017;Meng et al., 2019). MiR-490 was proved to have important effects in many fields. Previous reports found that miR-490 plays an essential role in tumors (Liu et al., 2015;Xu et al., 2015). MiR-490 can also inhibit the apoptosis of cells in the injured spinal cord and reduce the expression of inflammatory factors by targeting MK2 . However, the effects of miR-490 on sepsis-induced lung injury have not been reported.
Multidrug resistance-associated protein 4 (MRP4), a member of ATP-binding cassette transporters, acts as an energy-dependent transporter for cyclic nucleotides. The occurrence of sepsis ALI is closely related to the reduction in intracellular cyclic adenosine phosphate (cAMP) levels (Schlegel & Waschke 2014). MRP4 is a transmembrane protein that actively transports cAMP in cells (Yaneff et al., 2019). Therefore, MRP4 may have a profound regulatory effect on the dysfunction of vascular endothelial barrier in sepsis (Xia et al., 2019).
In this study, mice sepsis-induced ALI and PM-VECs models were established to study the effect of miR-490 on sepsis-induced ALI. The histopathology, injure score, Wet/Dry (W/D) ratio of lung tissue were assessed to verify that the model was successfully established. Furthermore, expression levels of inflammatory cytokines including TNF-α and IL-1β were also detected in ALI mice model. These results may provide new ideas for the treatment of infectious ALI.

Animals
32 C57BL/6 mice (5 days of age, 20-22 g, 16 males and 16 females) were purchased from Beijing Wei Tong Li Hua Experimental Animal Technology Co., Ltd., (Beijing, China; license: SCXK (Beijing] 2012-0001). Mice were fed in specific pathogen-free (SPF) animal rooms at 22±2°C, with a humidity of 40% and 12/12-h light/dark cycle. Mice had free access to chow and tap water. All experiments were approved by the Ethics Committee of Ruian People's Hospital and performed following the Guide for the Care and Use of Laboratory Animals (1996).

CLP mice model and groups
After one-week acclimatization, mice were randomly divided into the Sham group (n=8) and CLP model group (n=24). Mice underwent CLP surgery to establish the CLP-induced sepsis mice model as previously described Sureshbabu et al., 2018). Firstly, mice were anesthetized by 6% chloral hydrate (300 mg/kg, Sigma-Aldrich, St. Louis, MI, USA). Secondly, 2 cm long incision along the midline of the abdomen was performed to open the abdomen and expose the cecal. Thirdly, the cecal was ligated at 1/2 distance from its base of cecal, the cecal was punctured twice with an 18G needle, and a small amount of intestinal content was squeezed out. Finally, the bowel was sealed and the abdomen was closed using sterile sutures. After the operation, mice were injected subcutaneously with 0.9% saline (30 ml/kg) to be resuscitated. Sham group (n=8): mice received only laparotomy for cecal exploration without ligation and perforation. recorded.

Delivery of miR-490 in vivo
The miRNA reagents, miR-490 agomir and scrambled control (NC agomir) were purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China) and then mixed with linear polyethyleneimine (PEI) nanoparticles (Sigma-Aldrich, St. Louis, MO) as previously described (Morishita et al., 2015). CLP mice were further randomly divided into 3 groups 7 days before the CLP operation. CLP group (n=8): mice underwent routine CLP surgery; CLP+NC agomir (n=8): mice were intravenously injected with a total of 200 μL mixture containing 5 nM NC agomir via the tail vein, and then underwent routine modeling surgery; CLP+miR-490 agomir (n=8): mice were intravenously injected with a total of 200 μL mixture containing 5 nM miR-490 agomir via the tail vein, and then underwent routine modeling surgery. The mice were sacrificed and lungs were collected for further analysis.

Histological analysis
The lungs specimens were embedded in paraffin, sectioned, and stained using hematoxylin and eosin (H&E). The histological staining was examined under a DP73 Digital microscope (Olympus Corp., Tokyo, Japan). The degree of lung injury was estimated in a blinded manner.

Wet/Dry (W/D) ratio of lung tissue
After isolation of the lung tissue, the surface moisture was absorbed, then the wet mass (W) was weighed. The tissue was dried at 70°C for 48 h, then the dry mass (D) was weighed, and the lung W/D ratio was calculated.

BALF sample collection and measurements
The airway was washed with 10 ml physiological saline 3 times: 4 ml, 4 ml and 3 ml. The recovered solution was collected and centrifuged at 1,500 rpm for 10 min at 4°C, then the supernatant was collected and stored at -20°C. The total protein concentration was calculated using the BCA protein assay kit (Beyotime Biotechnology, China) according to the instructions. After centrifugation, BALF was resuspended with 50 ml 0.9% normal saline, and the number of cells in BALF was counted with a cell counter. The neutrophil number was assessed by ELISA kit (Denley Dragon Wellscan MK 3, Thermo, Vantaa, Finland) according to the manufacturer's protocol. Macrophage number was assessed using M-CSF ELISA Kit according to the manufacturer's protocol (Shanghai Renjie Biotechnology Co., LTD, China). Each sample was analyzed in triplicate.

Western blot
Total protein was isolated from MPVECs and quantified using a protein assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Equal amounts of protein (20 μg) were separated by 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore Corp., Billerica, MA, USA). Non-specific sites were blocked with 5% milk powder diluted in TBS with 0.05% Tween 20 (TBST). The membranes were incubated overnight at 4°C with rabbit anti-Bcl-2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-Cleaved caspase-3 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-MRP4 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After the membranes were washed repeatedly with TBST, they were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:5,000 dilution; Zhongshan Jinqiao Biological Technology Co., Ltd., Beijing, China). The blots were assessed by enhanced chemiluminescence. The expression levels of Pin1 and mTOR in esophageal epithelial tissue were quantified by densitometry using the Quantity One software (Bio-Rad, Hercules, CA, USA). The expression levels of Bcl-2, cleaved caspase-3, and MRP4 were normalized to GAPDH.

Enzyme-linked immunosorbent assay (ELISA)
The concentrations of TNF-α and IL-1β in mice BALF, lung tissue and MPVEC lysates were measured with ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instruction.

Apoptosis assay
Annexin-V FITC kit (Beijing BaosaiBiotechnology) was used to assess MPVECs apoptosis. Briefly, after MPVECs were treated as mentioned previously the cells were washed with PBS buffer twice, then 1×10 6 /mL cell suspension was prepared with 1× Binding Buffer. Annexin-V and nucleic acid dye were added to 100 ul of the suspension, mixed gently and incubated for 15 min. The cells were washed with 1× Binding Buffer once and the supernatant was removed. Then 5 ul PI were added to the cells, mixed and incubated for 15 min. Finally, 400 ul of 1× Binding Buffer was added to the mixture, incubated for 1 h and the samples were analyzed by flow cytometry.

Statistical analysis
All data were presented as means ± standard deviations. The differences between the groups were assessed using one-way Analysis Of Variance (ANOVA) with the Student-Newman-Keuls (SNK) test for posthoc analysis. All data were analyzed using SPSS 17.0 (IBM, Armonk, NY, USA). p<0.05 was considered statistically significant.

MiR-490 was downregulated in CLP-induced sepsis mice and the up-regulation of miR-490 alleviated sepsisinduced ALI
In order to study the role of miR-490 in ALI in sepsis mice, CLP was used to establish a mouse sepsis model. Compared to the sham group, miR-490 was significantly downregulated in CLP mice. However, the expression of miR-490 was significantly increased after the injection of miR-490 agomir when compared to that of NC agomir (Fig. 1A). As shown in Fig. 1B, compared to the sham group, the pulmonary tissues of mice in the sepsis group were significantly hyperemic, and alveolar collapse and alveolar wall thickening were present. However, these symptoms were relieved in comparison to the group injected with miR-490 agomir (Fig. 1B). Lung injury score was used to assess the extent of lung tissue damage, and overexpression of miR-490 significantly improved the histological damage caused by CLP surgery (Fig. 1C). Compared to the sham group, the W/D ratio of the lung tissue from mice in the sepsis group was increased significantly. However, overexpression of miR-490 significantly reduced the W/D ratio of the lung tissue from sepsis mice (Fig. 1D). Taken together, these results revealed that up-regulation of miR-490 reduced CLP-induced ALI in mice.

Overexpression of miR-490 decreased the expression of inflammatory cytokines in sepsis mice model
BALF and lung tissue were collected to examine the level of lung inflammatory cytokines in sepsis mice model. As shown in Fig. 2, compared to the sham group, the total protein concentration, cell count, neutrophils, macrophages, TNF-α and IL-1β were all increased in BALF from sepsis model mice. However, overexpression of miR-490 significantly decreased the total protein concentration, cell count, neutrophils, macrophages, TNF-α and IL-1β levels in BALF from sepsis model mice ( Fig. 2A, 2B, 2C, 2D, 2E). Similarly, in the lung tissues from sepsis model mice, the expression of TNF-α and IL-1β was increased significantly compared to the sham mice, and overexpression of miR-490 significantly reduced the levels of TNF-α and IL-1β (Fig. 2F). To sum up, overexpression of miR-490 significantly decreased the production of pro-inflammatory cytokines in sepsis model mice.

Overexpression of miR-490 reduced LPS-induced inflammation and apoptosis in PMVECs
In order to study the effect of miR-490 on PMVECs, mice PMVECs were stimulated with LPS to establish a cell model of ALI, and cells were transfected with miR-490 agomir. qRT-PCR results showed that, compared to the control group, LPS stimulation significantly reduced the levels of miR-490. Besides, the transfection with miR-490 agomir significantly enhanced miR-490 expression (Fig. 3A). Flow cytometry data showed that LPS could promote the apoptosis of MPVECs, and miR-490 agomir transfection could effectively decrease the apoptotic rate of MPVECs (Fig. 3B). Further analysis revealed that overexpression of miR-490 increased the protein level of Bcl-2 and decreased the protein level of cleaved caspase-3 compared to the transfection with NC agomir (Fig. 3C). Overexpression of miR-490 significantly reduced the protein levels of TNF-α and IL-1β (Fig. 3D).
In summary, overexpression of miR-490 reduced LPSinduced inflammation and apoptosis of PMVECs.

MRP4 was a potential downstream target of miR-490
To explore the potential mechanism of miR-490 action in ALI, we performed bioinformatics analysis using Tar-getScan (http://www.targetscan.org/vert_71/) to predict the downstream targets of miR-490. MRP4 was predicted as a direct target gene of miR-490, with putative binding sites (Fig. 4A). The dual-luciferase reporter gene experiment showed that in cells co-transfected with miR-490 mimics and MRP4-WT, the luciferase activity was significantly lower than that in case of transfection with miR-490 NC and MRP4-WT. However, there were no significant differences in the luciferase activity between cells cotransfected with MRP4-MUT+miR-490 and the MRP4-MUT+NC group (Fig. 4B). In addition, we measured the expression of MRP4 found that the expression level of MRP4 in LPS-treated cells and CLP group was higher than that in the corresponding control and sham group, respectively. However, the expression levels of MRP4 in LPS+miR-490 agomir group and CLP+miR-490 agomir group were significantly lower than that in LPS+NC agomir group and CLP+NC agomir group, respectively, indicating that miR-490 could negatively regulate the expression of MRP4 (Fig. 4C, 4D).

MiR-490 alleviates LPS-induced lung cell injury by regulating MRP4 expression
To demonstrate whether miR-490 can alleviate LPSinduced lung cell damage by regulating MRP4 expression, cells were transfected with recombinant adenovirus expressing MRP4. As shown in Fig. 5A, after the trans-fection the effects of miR-490 agomir on the expression levels of MRP4, Bcl-2 and cleaved caspase-3 were reversed. The transfection with recombinant adenovirus expressing MRP4 significantly increased the levels of TNF-α and IL-1β decreased by miR-490 agomir (Fig. 5B). Taken together, these results suggested that  miR-490 alleviated LPS-induced lung cell injury by regulating MRP4 expression.

DISCUSSION
Sepsis-induced ALI is a serious inflammatory disease of the lungs, accompanied by alveolar capillary inflammatory damage and inflammatory cytokines production (Miyashita et al., 2016). At present, the mortality rate of sepsis-ALI and ARDS has reached 40%, which is one of the main reasons of death in ICUs (Singh et al., 2014). Previous studies explored the pathogenesis and pathophysiology of sepsis-induced ALI in terms of genes and signaling pathways, but it is still not fully understood Meng et al., 2019). To provide new ideas for the treatment of sepsis-induced ALI, we thus studied the role and related mechanisms of miR-490 in sepsis-induced ALI. In the current study, we simultaneously established animal and cell models to investigate the role of miR-490 in ALI in vitro and in vivo and reveal its mechanism of action.
Firstly, we established a mice model of sepsis by CLP. Results showed that the lung injury score washigh, the histological examination revealed obvious congestion of the lungs, the collapse of the alveoli, thickening of the alveolar wall, and W/D results showed obvious pulmonary edema. These results were similar to previous studies, proving that a mouse model of sepsis was successfully established (Hamaguchi et al., 2015;Deng et al., 2018).
It is generally believed that the occurrence of ALI is related to the inflammatory cascade and neutrophil adhesion (Jia et al., 2016). TNF-α and IL-1β are key cytokines in the inflammatory response in the body, and also important factors leading to lung injury in sepsis. Controlling excessive inflammatory response is the key to treat ALI (Park et al., 2001;Lee et al., 2018). In the present study, the number of neutrophils, macrophages, and the levels of TNF-α and IL-1β were increased significantly in ALI mice. Liu and others  found that the level of TNF-α was markedly increased in the CLP group. Meng and others (Meng et al., 2019) also found that IL-1β level was increased following CLP. Furthermore, the total protein concentration and cell number were increased significantly, indicating that the recruitment of inflammatory cells in alveoli damages the alveolar epithelium and pulmonary vascular endothelial cells, thus increasing the exudation of alveolar proteins and contributing to the occurrence of ALI. Overexpression of miR-490 was achieved through intravenous injection of miR-490 agomir. In the present study, overexpression of miR-490 significantly alleviate the increase in the neutrophils, macrophages, TNF-α and IL-1β levels, the total protein concentration, and cell numbers caused by CLP. These results indicated that overexpression of miR-490 could effectively reduce the recruitment of inflammatory cells and alveolar exudation, and thus relieve the symptoms of ALI. Ju and others (Ju et al., 2018) found that microRNA-27a could inhibit inflammation and alleviate ALI in mice. Suo and others (Suo et al., 2018) reported that miRNA-1246 could suppress ALI-induced inflammation in mice. Moreover, Xie and others (Xie et al., 2018) found that miR-34b-5p could inhibit ALI inflammation.
Secondly, LPS was used to stimulate cells to establish cell models and investigate the effect of miR-490 in vitro. Similarly to animal experiments, the levels of TNF-α and IL-1β in LPS -treated PMVECs were increased, and overexpression of miR-490 could inhibit these effects. What is more, LPS decreased the levels of Bcl-2 and elevated the levels of cleaved caspase-3, which led to a high rate of apoptosis in PMVECs. However, overexpression of miR-490 effectively relieved the apoptosis of PMVECs. It is well-known that Bcl-2 and caspase-3 play an irreplaceable role in apoptosis (Liu et al., 2020;Yuan & Zhang 2020). To sum up, this study indicated that the upregulation of miR-490 could affect the progression of ALI in vivo and in vitro. However, the mechanism of action remained unknown. When studying the mechanism of miR-490 in ALI, we speculated that miR-490 might target specific molecules or regulate downstream genes to regulate inflammation and apoptosis-related pathways during ALI progression. MiR-490-3p could inhibit esophageal squamous cell carcinoma via targeting HMGA2 (Kang et al., 2018). Overexpression of miR-490-5p inhibited the metastasis of hepatocellular carcinoma cells via targeting E2F2 and ECT2 (Fang et al., 2018). Downregulation of miR-155 could inhibit lung apoptosis and inflammation via SIRT1 (Ju et al., 2018). The downregulation of miR-1246 could decrease cells apoptosis, IL-1b, and neutrophil infiltration by targeting ACE2 (Fang et al., 2017). It was also reported that miR-539-5p could alleviate ALI by targeting ROCK1 (Meng et al., 2019). Here, the dual-luciferase assay indicated that miR-490 targeted MRP4. Moreover, overexpression of miR-490 inhibited the expression of MRP4 in vitro and in vivo.
The protective effect of MRP4 inhibition on ALI caused by sepsis may be exerted by reducing pulmonary vascular permeability and reducing inflammation. MRP4 can regulate the permeability of endothelial cells by regulating the downstream molecules -prostaglandins. It was reported that MRP4 inhibitor MK571 could reduce lung damage in rats with sepsis (Zheng et al., 2016). In order to further investigate the mechanism of action, MRP4 was successfully overexpressed, and the results showed that overexpression of MRP4 could reverse the inhibitory effect of miR-490 overexpression on cell apoptosis and increase the levels of inflammatory factors. Xia, et al., reported that inhibition of MRP4 could decrease serum inflammatory factors levels in BALF of septic mice, and the protective effect of MRP4 inhibition on sepsisinduced ALI might be associated with the elevation of cAMP levels (Xia et al., 2019). cAMP is an important second messenger in cells, which can inhibit the damage dealt by inflammatory factors to the pulmonary vascular endothelium by increasing the intracellular cAMP level. The inhibition of MRP4 was also found to decrease inflammation significantly (Leite et al., 2007). However, some limitations in the present study may exist. First, there are many cell types involved in ALI, such as endothelial cells, epithelial cells and inflammatory cells. However, the focus of this study was only on endothelial cells that proved to play an important role in ALI progression. Since MRP4 exists in various cell types and regulates various biological processes, it is necessary to further determine the regulatory effects of MRP4 on other cell types. Second, miR-490 may produce its biological effects by regulating other proteins, such as Golgi intermediate compartment protein 3 (ERGIC3). Besides, our study did not involve MRP4 silencing experiment, to prove the biological role of miR-490-MRP4 interaction.
In conclusion, miR-490 relieved the sepsis-induced ALI via downregulating the expression of MRP4, which indicates that miR-490 might act as a potential therapeutic target for sepsis-induced ALI.