miR-146b-3p regulates PI3K/AKT signaling pathway in septic mice with acute respiratory distress syndrome CURRENT STATUS: UNDER REVIEW

Background: This study aimed to explore the effect of miR-146b-3p on acute respiratory distress syndrome in septic mice by regulating PI3K/AKT signaling pathway. Methods: Seventy C57BL/6 mice were divided into normal group ( n = 10) and modeling group ( n = 60, mice for constructing septic mice models with acute respiratory distress syndrome). Model mice were subdivided into model group (without any treatment), negative control (NC) mimic group (injection with miRNA NC), miR-146b-3p mimic group (injection with miR-146b-3p mimic), si-NC group (injection with PI3Kγ siRNA NC), si-PI3Kγ group (injection with PI3Kγ interference sequence), and miR-146b-3p mimic + oe-PI3Kγ group (injection with miR-146b-3p mimic + PI3Kγ overexpression plasmid). Dual-luciferase reporter assay was conducted to determine the target relationship between miR-146b-3p and PI3Kγ. Wet weight/dry weight (W/D) ratio of the left lung was measured. Hematoxylin and eosin stain was used to detect the pathological change of mouse lung. ELISA was employed to measure serum interleukin (IL) -1β and IL-18 levels. miR-146b-3p and PI3Kγ expressions were detected by qRT-PCR. PI3Kγ, AKT, NLRP3, apoptosis-associated speck-like protein caspase recruitment domain (ASC) and Caspase-1 protein expressions were detected by Western blotting. Results: miR-146b-3p negatively regulated PI3Kγ. The lung tissues in other groups compared with normal group had down-regulated miR-146b-3p, up-regulated PI3Kγ, p-AKT, ASC, NLRP3 and Caspase-1 proteins, higher W/D ratio, and more serum IL-1β and IL-18 (all P < 0.05). All indicators in miR-146b-3p mimic group and si-PI3Kγ group were significantly improved as compared to model group (all P < 0.05).


Background
Acute respiratory distress syndrome (ARDS) is a non-cardiogenic pulmonary edema caused by the accumulation of extravascular lung water as well as a major complication of severe sepsis and septic 3 shock [1]. ARDS is characterized by hypoxemia, pulmonary edema, and significant respiratory failure over time, which leads to multiple organ failure and high mortality (up to 60%) [2][3][4][5].
Lipopolysaccharide (LPS) is the main method to construct sepsis-induced ARDS model, because it can activate inflammatory cells and release inflammatory factors interleukin-1β (IL-1β) and interleukin-18 (IL-18) [6]. NLRP3 inflammasome has vital function in the pathological process of ARDS. NLRP3 inflammasome as a protein complex contains pro-caspase-1 and apoptosis-associated speck-like protein caspase recruitment domain (ASC); active caspase-1 stimulates IL-1β and IL-18, causing the release of these active cytokines [7].
As a complex pathological process, ARDS is regulated by transcription factors as well as miRNA. A miRNA is a small endogenous non-coding RNA molecule that includes approximately [18][19][20][21][22][23][24] nucleotides. It usually complements sites in the 3' untranslated region of the target messenger RNA for inhibiting the expression of post-transcriptional gene [8]. Several studies have found that multiple miRNAs affect the occurrence of ARDS, and microRNA-122 has relations with the mortality of ARDS and acute liver injury [9]; microRNA-211 induced by NF-κB inhibits the function of macrophages releasing IL-10 in LPS-induced ARDS rats [10]. Down-regulation of microRNA-494 alleviates lung injuries in sepsis-related ARDS by regulating NQO1-Nrf2 signaling pathway [11]. microRNA-23a-5p can be used as a potential biomarker for early sepsis induced ARDS [12]. miR-146b mainly functions as a regulatory factor of inflammation and cancer [13]; reports about miR-146b are mostly related to cancer, but little is about its effect on ARDS [14]. AKT1 gene, known as protein kinase Bα, is a key member in AKT family. Moreover, AKT is generally recognized as a key factor in PI3K (phosphatidylinositol 3-kinase) /AKT pathway and acts as a crucial role in cell differentiation, proliferation, metabolism, apoptosis, protein synthesis and transcription [11]. Previous studies have indicated that ARDS can be treated by the inhibition of PI3K/AKT signaling pathway [15][16][17][18].
A target relationship between miR-146b-3p and PI3Kγ was found by bioinformatics prediction. We speculated that miR-146b-3p, by targeted down-regulation of PI3Kγ expression, might repress PI3K/AKT signaling pathway, thus improving ARDS in septic mice.

Grouping and treating
Seventy two-week-old healthy male C57BL/6 mice of clean grade (purchased from the Laboratory Animal Center of Wenzhou Medical University), weighing 35±5 g, were divided into normal group (n = 10) and modeling group (n = 60). Mice in modeling group were used to construct septic mice models with ARDS [19]. Mice were anesthetized by intraperitoneal administration of 50 mg/kg pentobarbital sodium before exposing their trachea and right internal jugular vein; 5 mg/kg LPS (escherichia coli 0111:B4) were dripped once into 50 μL sterile phosphate buffered saline (PBS). A total of 3×10 7 PFU Ad-omentin or Ad-βgal was injected into the internal jugular vein of mice 3 days before installing the airway with LPS or gas (PBS). All mice in normal group survived, and 18 mice in modeling group died 24 h later. Model mice were subdivided into 6 groups: model group (without any treatment), negative control (NC) mimic group (injection with miRNA NC), miR-146b-3p mimic group (injection with miR-146b-3p mimic), si-NC group (injection with PI3Kγ siRNA NC), si-PI3Kγ group (injection with PI3Kγ interference sequence), and miR-146b-3p mimic + oe-PI3Kγ group (injection with miR-146b-3p mimic + PI3Kγ overexpression plasmid). Sequences and plasmids (Suzhou Jima Gene Co. Ltd., China) of 100 ng were intraperitoneally injected in mice once on alternate days for 3 times. Mice in each group were anesthetized using 0.1 ml/10 g 0.3% pentobarbital sodium solution after treatment. Then the eyeball was removed for blood collection to make mice die due to excessive blood loss under anesthesia, and lung tissue specimens were reserved. The lung tissues and venous blood of 5 mice in each group were used for detection, and the remaining tissues were stored in liquid nitrogen. This study was approved by the Ethics Committee of The First People's Hospital of Wenling.

Dual-luciferase reporter system
The binding site of miR-146b-3p and PI3Kγ was analyzed by bioinformatics prediction website (www.targetscan.org). Next, the target relationship between miR-146b-3p and PI3Kγ was verified by dual-luciferase reporter system. Dual-luciferase reporter gene vector of target gene PI3K and mutant at the binding site of miR-146b-3p and PI3Kγ, PGL3-PI3Kγ wt and PGL3-PI3Kγ mut, were constructed, respectively. Rellina plasmid and the two reporter plasmids with miR-146b-3p plasmid and NC plasmid respectively were co-transfected into HEK293T cells. Dual-luciferase reporter assay was performed after 24 h cell transfection. Cells in each group were lysed and centrifuged at 12,000 rpm for 1 min. The precipitation was discarded and the supernatant was collected. Luciferase activity was measured according to the instruction of dual-luciferase reporter kit (Promega, USA). Lysed cell samples were pipetted into eppendorf tubes. The 100 μL firefly luciferase working solution were added into every 10 μL samples to determine firefly luciferase activity followed by the addition of 100 μL ranilla luciferase working solution to determine ranilla luciferase activity. Relative luciferase activity = firefly luciferase activity / ranilla luciferase activity. -ΔCt (control group) . miR-146b-3p and PI3Kγ took U6 and GAPDH as the internal references, respectively.

qRT-PCR
2 -ΔΔCt showed the relative expression level of a target gene. Primers were shown in Table 1. Table 1 qRT-PCR primer sequence.

Western blot
We extracted total protein by using RIPA lysis buffer containing PMSF (R0010, Solarbio Then images were exposed by a gel imager. Images were taken using Bio-Rad image analysis system (BIO-RAD, USA) and analyzed using ImageJ software. Relative protein expression = gray value of the corresponding protein band / gray value of GAPDH protein band.

Wet weight/dry weight ratio
The left lung of the mouse was taken out by thoracotomy, and the blood on the lung surface was sucked dry by filter papers. Then wet weight was weighed out. After the left lung was baked in the oven at 80 °C for 48 h to achieve constant weight, the dry weight was weighed out. Wet weight/dry weight (W/D) ratio and lung water content were calculated to reflect the severity of pulmonary edema. W/D ratio of the lung = wet weight of the lung / dry weight of the lung × 100%.

Hematoxylin and eosin stain
The

Statistical analysis
All data were analyzed by SPSS21.0 statistical software. The measurement data were expressed as mean ± standard deviation. Comparison among groups was performed by one-way analysis of variance, and pairwise comparison of the mean among groups was carried out by Tukey post-hoc test.
P < 0.05 indicated a statistically significant difference.

miR-146b-3p, PI3Kγ, AKT, p-AKT expressions in the lung tissue
We detected the expressions of lung tissue-related factors in each group to further confirm the regulation relationship between miR-146b-3p and PI3K/AKT signaling pathway (Fig. 2). The lung tissue in the rest groups compared with normal group had lower miR-146b-3p expression and higher PI3Kγ expression (both P < 0.05). Compared with model group, NC mimic group, si-NC group, and miR-146b-3p mimic + oe-PI3Kγ group had no significant differences in gene expressions (P > 0.05), and miR-146b-3p mimic group and si-PI3Kγ group had lower PI3Kγ and p-AKT protein expressions (both P < 0.05). miR-146b-3p mimic + oe-PI3K group had higher PI3Kγ and p-AKT protein expressions as compared to miR-146b-3p mimic group (both P < 0.05).

Pathological change of mouse lung
Pathological change of mouse lung was detected by hematoxylin and eosin staining (Fig. 3). Mice in normal group had regular lung structure and no significant pathological injury. Mice in other groups had various degrees of inflammatory cell infiltration in the pulmonary alveoli and mesenchyme, effusion in the cavity, obvious thickening alveolar septum, partial collapsed alveoli, atelectasis, hyaline membrane, and damaged alveoli. Lung injuries in miR-146b-3p mimic group and si-PI3Kγ group were significantly improved than model group, NC mimic group, si-NC group, and miR-146b-3p mimic + oe-PI3Kγ group.

Wet weight/dry weight ratio of the lung
W/D ratio of the lung was significantly higher in other groups than normal group (P < 0.05). Compared with model group, NC mimic group, si-NC group, and miR-146b-3p mimic + oe-PI3Kγ group had no difference in W/D ratio (P > 0.05), and W/D ratio was significantly lower in miR-146b-3p mimic group and si-PI3Kγ group (P < 0.05). W/D ratio was significantly higher in miR-146b-3p mimic + oe-PI3Kγ group than in miR-146b-3p mimic group (P < 0.05, Fig. 4).

Levels of IL-1β and IL-18 in serum
Levels of IL-1β and IL-18 in serum were detected by ELISA (Fig. 5). Levels of IL-1β and IL-18 were significantly higher in other groups than in normal group (both P < 0.05). Compared with model group, NC mimic group, si-NC group, and miR-146b-3p mimic + oe-PI3Kγ group had no difference in levels of IL-1β and IL-18 (both P > 0.05), and miR-146b-3p mimic group and si-PI3Kγ group had significantly lower levels of IL-1β and IL-18 (both P < 0.05). Levels of IL-1β and IL-18 were significantly higher in miR-146b-3p mimic + oe-PI3Kγ group than in miR-146b-3p mimic group (both P < 0.05).

Discussion
Sepsis and acute respiratory distress syndrome (ARDS) are two types of syndromes needing intensive care with multiple characteristics. Both are common and highly lethal and have significant negative effects on survivors [20]. Sepsis-induced ARDS has an estimated incidence of over 40% and is the leading cause of ARDS death [21]. ARDS is a life-threatening complication featured by diffuse alveolar damage [22]. The incidence of ARDS varies widely, ranging from 1.5 patients per 100,000 people to nearly 79 patients per 100,000 people; European countries report lower incidence than the United States; mortality also varies due to the severity, age, and underlying medical conditions [23][24][25][26][27]. No monotherapy has been found to alter the latent pathological process of ARDS to date. The molecular mechanism for preventing the onset of ARDS is unclear and needs to be solved urgently [28].
In this study, down-regulated miR-146b-3p was found in sepsis-induced ARDS. Literatures revealed the activated PI3K/AKT signaling pathway in ARDS [16,29]. In this study, we used PI3K interference sequence to treat C57BL/6 model mice and found that after PI3K/AKT signaling pathway was blocked, there were lower protein expressions of PI3Kγ, AKT, ASC, NLRP3 and Caspase-1, W/D ratio and serum IL-1β and IL-18 levels and improved inflammation in the lung tissues of mice. The results showed that inhibition of PI3K/AKT signaling pathway could improve lung injuries caused by ARDS in septic mice.
To further explore the upstream regulatory mechanism of PI3K/AKT signaling pathway, we verified the target relationship between PI3Kγ and miR-146b-3p through bioinformatics prediction website and confirmed through dual-luciferase reporter assay that miR-146b-3p negatively regulated PI3Kγ. Model mice were injected with miR-146b-3p inhibitor + oe-PI3Kγ and miR-146b-3p mimic. The results indicated that miR-146b-3p overexpression improved lung injuries caused by ARDS in septic mice. levels, and improving tissue inflammation, which was consistent with previous reports on ARDS in septic mice [30][31][32][33][34].

Conclusions
In this study, we demonstrate that miR-146b-3p, by targeting PI3Kγ gene, mediates PI3K signaling pathway to inhibit the recovery of lung injury in model mice. We further clarify the development mechanism of ARDS in septic mice, which lays a theoretical foundation for the clinical treatment of ARDS in septic mice. However, the association between miR-146b-3p and ARDS in septic mice has not been fully explained to date; the molecular mechanism by which PI3K/AKT signaling pathway regulates inflammasome NLRP3, ASC and Caspase-1 has not been fully explored; the targeted regulatory network of miR-146b-3p in sepsis-induced ARDS mice is still unclear.

Ethics approval
This study was approved by the Ethics Committee of The First People's Hospital of Wenling.

Not applicable
Availability of data and material: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JQZ and XHJ contributed to data collection and data statistics. MPD collected data. YL and JFZ contributed to data analysis, manuscript concept and editing, and manuscript guidance. All authors read and approved the final manuscript.  Wet weight/dry weight ratio of the lung. Compared with normal group, *P < 0.05; compared with model group, #P < 0.05; compared with NC mimic group, %P < 0.05; compared with miR-146b-3p mimic group, &P < 0.05; compared with si-NC group, $P < 0.05; compared with si-PI3Kγ group, @P < 0.05. W/D Wet weight/dry weight, NC negative control NC3Rs ARRIVE Guidelines Checklist.pdf