# Inhibition of miR-29b-1-5p Attenuates Inflammatory Response and Pulmonary Fibrosis in LPS-Induced Acute Lung Injury by Regulating RTN4 Expression

Authors: Jieqiong Wang; Ming Chen; Weihua Xu; Lu Shou; Xiaosheng Jin; Xianrong Xu; Feihua Huang
Journal: Evidence-Based Complementary and Alternative Medicine
 (2022)

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## Abstract

Objective. Acute lung injury (ALI) is a severe respiratory disorder causing alveolar-capillary barrier, leading to a high rate of morbidity and death in critically ill individuals. microRNAs (miRNAs)-mediated mechanism in the pathogenesis of ALI has attracted much interest. Herein, we attempt to characterize a candidate miRNA and its downstream target that is linked to the pathogenesis of ALI. Methods. LPS-conditioned MH-S cells were treated with miR-29a-1-5p mimic, inhibitor, and RNT4 expression vector, and the ALI animal model was injected with agomir and antagomir of miR-29b-1-5p and RNT4 expression vector, in which the pro-inflammatory cytokine production, cell viability and apoptosis, myeloperoxidase (MPO) activity, wet/dry (W/D) ratio, and expression of TGF-β1, α-smooth muscle actin (α-SMA), E-cadherin, and vimentin were examined. miR-29a-1-5p inhibition of RTN4 translation was confirmed by luciferase activity assays. Results. An elevated miR-29a-1-5p expression was demonstrated in LPS-conditioned MH-S cells. miR-29a-1-5p inhibitor transfection attenuated the production of pro-inflammatory cytokines and MH-S cell viability but enhanced the apoptosis. miR-29a-1-5p inhibition of RTN4 translation was demonstrated in the setting of LPS-induced ALI. LPS-induced murine models demonstrated upregulated miR-29a-1-5p. Intravenous injection of miR-29b-1-5p agomir attenuated mouse lung injury and pulmonary fibrosis. RTN4 overexpression resisting to miR-29a-1-5p overexpression was demonstrated in LPS-induced murine models. Conclusion. The findings obtained from the study that disturbing the action of miR-29a-1-5p may be a novel therapeutic strategy for preventing ALI.


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## Body

## 1. Introduction

As a lethal complication resulting from distant organ dysfunction or acute injury such as shock and sepsis, acute lung injury (ALI) is described as the major cause of Intensive Care Unit (ICU) death across the world [1]. Moreover, it has been reported that inflammatory response is the major pathological change of ALI, leading to accumulated inflammatory cells, interstitial edema, and disrupted epithelial integrity [2]. Lipopolysaccharide (LPS) is known as a leading element of the outer membrane in Gram-negative bacteria, which serves as a key factor for the occurrence of ALI [3, 4]. Owing to limited treatment options and complex pathogenesis, ALI is considered a significant contributor to global morbidity and mortality concerning acute respiratory failure [5, 6]. Although there are great advancements in developing candidate therapy strategies for ALI, its mortality rate remains from 22% to 40% [7]. Therefore, it is important to further investigate new prevention strategies and therapeutic methods against ALI.microRNAs (miRNAs) represent a group of small noncoding RNA and have the ability to modulate gene expression via targeting mRNA for early degradation or inhibition of its translation. In light of miRNA control of inflammatory-immune responses and cell-cell interactions from previous evidence [8], miRNAs are reported to be involved in the pathological processes of various lung diseases, including ALI [9]. Dysregulation of miR-29 family occurs in several human cancers, such as breast cancer [10], osteosarcoma [11], and bladder urothelial cancer [12]. Recently, upregulated miR-29b-1-5p was observed in hearts following I/R injury and in cardiomyocytes following hydrogen peroxide treatment [13]. The bioinformatics prediction shows RTN4 (encoding Nogo protein) is a putative target gene of miR-29b-1-5p. RTN4, also known as neurite outgrowth inhibitor, consists of three different splice variants (termed RTN4-A, RTN4-B, and RTN4-C) through different splicing [14]. RTN4-A could inhibit the migration and invasion ability of human malignant glioma cells [15]. A murine study has reported that the RTN4-B overexpression ameliorates lung injury, alveolar protein exudation, and neutrophil infiltration in LPS-induced ALI mice, suggesting protective effects of RTN4-B against ALI [16]. In this study, we propose a prevailing hypothesis that high miR-29b-1-5p expression contributes to the development of ALI by negatively regulating the RTN4. To prove this hypothesis, we examined the viability and apoptosis of LPS-induced murine alveolar macrophages MH-S, as well as the release of pro-inflammatory cytokines, lung injury, and pulmonary fibrosis LPS-induced murine models.


## 2. Materials and Methods

### 2.1. In Vitro ALI Models

The murine alveolar macrophages (MH-S) were purchased from Beijing Union Cell Institute (Beijing, China) and then maintained in the medium containing sodium bicarbonate (1.59/L), fetal bovine serum (FBS, 15%), and glutamine (2 mL). To condition the ALI cell model, MH-S cells were harvested in 100μg/mL LPS for 18 h. Subsequently, LPS-conditioned MH-S cells were treated with miR-29a-1-5p mimic, inhibitor, and RNT4 expression vector (GenePharma, Shanghai, China) using Lipofectamine 3000 reagents (Invitrogen, USA) as guided by the standard protocol provided by the manufacturer.


### 2.2. In Vivo ALI Models

A total of 74 C57BL/6J male mice, aged 7 weeks, were used to establish ALI murine models by intratracheal drip of 7.5 mg/kg LPS into the lungs of mice as described previously [7]. A longitudinal incision with about 0.5 cm in length was made in the neck for trachea exposure, and a mixture of LPS solution and 300 μl of sterile saline solution was injected into exposed trachea. Finally, we obtained 65 ALI murine models. At 6 h after modeling, 54 mice were injected with agomir and antagomir of miR-29b-1-5p, and RNT4 expression vector (GenePharma, Shanghai, China) via caudal vein. Among the remaining 11 LPS-induced ALI mice, 9 were served as control and 2 were spared. After 48 hours, experimental mice underwent tracheal intubation and bronchoalveolar lavage using 1.2 mL phosphate-buffered saline (PBS) three times to collect bronchoalveolar lavage fluid (BALF). At last, mice were euthanized by cervical dislocation. Animal experiments were carried out with the approval of the Institutional Animal Care and Use Committee of TongDe Hospital of Zhejiang Province.


### 2.3. Luciferase Assays

The wild-type RTN4 mRNA 3′UTR containing the binding sites of miR-29b-1-5p and the mutated one were cloned into the pGL3-reporter vectors (Sangon, Shanghai, China), respectively. Well-designed reporter vectors pGL3-RTN4-Wt and pGL3-RTN4-Mut with miR-29b-1-5p mimic were delivered into HEK293T cells (Beinuo, Shanghai, China). The dual-luciferase reporter assay system kit (K801-200, BioVision, USA) was utilized to determine the luminescence of firefly luciferase.


### 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA kit was used to measure levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant of mouse alveolar macrophages MH-S and of LPS-treated mouse alveolar lavage fluid.


### 2.5. Myeloperoxidase Activity Assays

Myeloperoxidase (MPO) is the functional and activating marker of neutrophils, and its level and activity change represent the function and active state of neutrophil polymorphonuclear leukocytes (PMN). The lung tissues were collected to measure MPO activity according to the manufacturer’s instructions of the MPO Activity Assay Kit (K747-100, BioVision (Milpitas, CA, USA).


### 2.6. Cell Viability Assays

MH-S cells were harvested, and their viability at 0, 24, 48, and 72 h was evaluated by another incubation with 10μl CCK-8 solution for 2 h. The optical value, also absorbance, was read at 450 nm, with growth curves plotted.


### 2.7. Annexin V-FITC/PI-Labeled Flow Cytometry

Annexin V-FITC/PI double staining was performed to examine cell apoptosis. The MH-S cell suspension (100µl, 4 × 105 cells) was incubated with 10 μL Annexin V-FITC and 5 μL PI without light exposure at room temperature for 15 min. The flow cytometer (6HT, Wuhan Cellwar Biotechnology Co., Ltd., Wuhan, China) was employed to measure the apoptosis.


### 2.8. Wet Weight/Dry Weight (W/D) Ratio

The left lung of mice without bronchoalveolar lavage was taken out following thoracotomy. After sucking the blood on the lung surface by filter paper, the lung was weighed to show the wet weight. With regard to measurement of the dry weight, the lung was maintained in an oven at 80°C for 48 h. The degree of pulmonary edema was evaluated by the dry/wet ratio (W/D).


### 2.9. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). For quantitation of miR-29b-1-5, total RNA was reverse-transcribed into cDNA using the miRNA first-strand cDNA synthesis (Tailing reaction) kit (B532451-0020, Shanghai Sangon Biotechnology Co. Ltd., China). For the quantitation of mRNA, total RNA was reverse-transcribed into cDNA using the kit (Takara, Japan). The qRT-PCR was conducted using the SYBR® Premix Ex TaqTM II kit (Takara) on the ABI7500 instrument (ABI, USA). The other primer sequences (Table1) were synthetized by Shanghai Sangon Biotechnology Co., Ltd. The Ct value was recorded, and the relative expression was calculated using the 2−ΔΔCt method, with GAPDH or U6 used to normalization.Table 1
Primer sequences for qRT-PCR.
TargetPrimermiR-29b-1-5p5′-GCTGGTTTCATATGGTGGTTTA-3′RTN4Forward: 5′-CTCCTCTGGTCTCGTCCTC-3′Reverse: 5′-GTCCTCGTCCTCCTCTTCC-3′TGF-β1Forward: 5′-AAACGGAAGCGCATCGAA-3′Reverse: 5′-GGGACTGGCGAGCCTTAGTT-3′α-SMAForward: 5′-CAGGGAGTAATGGTTGGAATG-3′Reverse: 5′-ATCGGATACTTCAGCGTCAG-3′E-CadherinForward: 5′-CCCACCACGTACAAGGGTC-3′Reverse: 5′-CTGGGGTATTGGG GGCATC-3′VimentinForward: 5′-GACAATGCGTCTCTGGCACGTCTT-3′Reverse: 5′-TCCTCCGCCTCCTGCAGGTTCTT-3′GAPDHForward: 5′-TTAGCACCCCTGGCCAAGG-3′Reverse: 5′-CTTACTCCTTGGAGGCCATG-3′


### 2.10. Western Blot Analysis

The total protein was extracted and then loaded into the wells added with 10% SDS-PAGE. After membrane wet transfer, immunoreaction was performed using anti-RTN4A/B antibody (ab47085), anti-Akt antibody (ab81283), anti-p-Akt antibody (ab38449), anti-ERK1/2 antibody (ab17942), anti-p-ERK1/2 antibody (ab223500), anti-TGF-β1 antibody (ab92486), anti-α-SMA antibody (ab5694), anti-E-cadherin antibody (ab1416), antivimentin antibody (ab92547), and anti-β-actin antibody (ab8226). All antibodies were purchased from Abcam Inc. (Cambridge, UK). Immunoreactive blots were visualized and quantified using a gel documentation system (Bio-Rad Quantity One Software v4.6.2, USA).


### 2.11. Statistical Analysis

Statistical comparisons including unpairedt-test, one-way analysis of variance, and repeated measurement analysis of variance and figure creation were carried out by GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA), with P<0.05 showing statistical significance. All data were summarized by mean ± standard deviation.


## 2.1. In Vitro ALI Models

The murine alveolar macrophages (MH-S) were purchased from Beijing Union Cell Institute (Beijing, China) and then maintained in the medium containing sodium bicarbonate (1.59/L), fetal bovine serum (FBS, 15%), and glutamine (2 mL). To condition the ALI cell model, MH-S cells were harvested in 100μg/mL LPS for 18 h. Subsequently, LPS-conditioned MH-S cells were treated with miR-29a-1-5p mimic, inhibitor, and RNT4 expression vector (GenePharma, Shanghai, China) using Lipofectamine 3000 reagents (Invitrogen, USA) as guided by the standard protocol provided by the manufacturer.


## 2.2. In Vivo ALI Models

A total of 74 C57BL/6J male mice, aged 7 weeks, were used to establish ALI murine models by intratracheal drip of 7.5 mg/kg LPS into the lungs of mice as described previously [7]. A longitudinal incision with about 0.5 cm in length was made in the neck for trachea exposure, and a mixture of LPS solution and 300 μl of sterile saline solution was injected into exposed trachea. Finally, we obtained 65 ALI murine models. At 6 h after modeling, 54 mice were injected with agomir and antagomir of miR-29b-1-5p, and RNT4 expression vector (GenePharma, Shanghai, China) via caudal vein. Among the remaining 11 LPS-induced ALI mice, 9 were served as control and 2 were spared. After 48 hours, experimental mice underwent tracheal intubation and bronchoalveolar lavage using 1.2 mL phosphate-buffered saline (PBS) three times to collect bronchoalveolar lavage fluid (BALF). At last, mice were euthanized by cervical dislocation. Animal experiments were carried out with the approval of the Institutional Animal Care and Use Committee of TongDe Hospital of Zhejiang Province.


## 2.3. Luciferase Assays

The wild-type RTN4 mRNA 3′UTR containing the binding sites of miR-29b-1-5p and the mutated one were cloned into the pGL3-reporter vectors (Sangon, Shanghai, China), respectively. Well-designed reporter vectors pGL3-RTN4-Wt and pGL3-RTN4-Mut with miR-29b-1-5p mimic were delivered into HEK293T cells (Beinuo, Shanghai, China). The dual-luciferase reporter assay system kit (K801-200, BioVision, USA) was utilized to determine the luminescence of firefly luciferase.


## 2.4. Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA kit was used to measure levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant of mouse alveolar macrophages MH-S and of LPS-treated mouse alveolar lavage fluid.


## 2.5. Myeloperoxidase Activity Assays

Myeloperoxidase (MPO) is the functional and activating marker of neutrophils, and its level and activity change represent the function and active state of neutrophil polymorphonuclear leukocytes (PMN). The lung tissues were collected to measure MPO activity according to the manufacturer’s instructions of the MPO Activity Assay Kit (K747-100, BioVision (Milpitas, CA, USA).


## 2.6. Cell Viability Assays

MH-S cells were harvested, and their viability at 0, 24, 48, and 72 h was evaluated by another incubation with 10μl CCK-8 solution for 2 h. The optical value, also absorbance, was read at 450 nm, with growth curves plotted.


## 2.7. Annexin V-FITC/PI-Labeled Flow Cytometry

Annexin V-FITC/PI double staining was performed to examine cell apoptosis. The MH-S cell suspension (100µl, 4 × 105 cells) was incubated with 10 μL Annexin V-FITC and 5 μL PI without light exposure at room temperature for 15 min. The flow cytometer (6HT, Wuhan Cellwar Biotechnology Co., Ltd., Wuhan, China) was employed to measure the apoptosis.


## 2.8. Wet Weight/Dry Weight (W/D) Ratio

The left lung of mice without bronchoalveolar lavage was taken out following thoracotomy. After sucking the blood on the lung surface by filter paper, the lung was weighed to show the wet weight. With regard to measurement of the dry weight, the lung was maintained in an oven at 80°C for 48 h. The degree of pulmonary edema was evaluated by the dry/wet ratio (W/D).


## 2.9. RNA Isolation and Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA). For quantitation of miR-29b-1-5, total RNA was reverse-transcribed into cDNA using the miRNA first-strand cDNA synthesis (Tailing reaction) kit (B532451-0020, Shanghai Sangon Biotechnology Co. Ltd., China). For the quantitation of mRNA, total RNA was reverse-transcribed into cDNA using the kit (Takara, Japan). The qRT-PCR was conducted using the SYBR® Premix Ex TaqTM II kit (Takara) on the ABI7500 instrument (ABI, USA). The other primer sequences (Table1) were synthetized by Shanghai Sangon Biotechnology Co., Ltd. The Ct value was recorded, and the relative expression was calculated using the 2−ΔΔCt method, with GAPDH or U6 used to normalization.Table 1
Primer sequences for qRT-PCR.
TargetPrimermiR-29b-1-5p5′-GCTGGTTTCATATGGTGGTTTA-3′RTN4Forward: 5′-CTCCTCTGGTCTCGTCCTC-3′Reverse: 5′-GTCCTCGTCCTCCTCTTCC-3′TGF-β1Forward: 5′-AAACGGAAGCGCATCGAA-3′Reverse: 5′-GGGACTGGCGAGCCTTAGTT-3′α-SMAForward: 5′-CAGGGAGTAATGGTTGGAATG-3′Reverse: 5′-ATCGGATACTTCAGCGTCAG-3′E-CadherinForward: 5′-CCCACCACGTACAAGGGTC-3′Reverse: 5′-CTGGGGTATTGGG GGCATC-3′VimentinForward: 5′-GACAATGCGTCTCTGGCACGTCTT-3′Reverse: 5′-TCCTCCGCCTCCTGCAGGTTCTT-3′GAPDHForward: 5′-TTAGCACCCCTGGCCAAGG-3′Reverse: 5′-CTTACTCCTTGGAGGCCATG-3′


## 2.10. Western Blot Analysis

The total protein was extracted and then loaded into the wells added with 10% SDS-PAGE. After membrane wet transfer, immunoreaction was performed using anti-RTN4A/B antibody (ab47085), anti-Akt antibody (ab81283), anti-p-Akt antibody (ab38449), anti-ERK1/2 antibody (ab17942), anti-p-ERK1/2 antibody (ab223500), anti-TGF-β1 antibody (ab92486), anti-α-SMA antibody (ab5694), anti-E-cadherin antibody (ab1416), antivimentin antibody (ab92547), and anti-β-actin antibody (ab8226). All antibodies were purchased from Abcam Inc. (Cambridge, UK). Immunoreactive blots were visualized and quantified using a gel documentation system (Bio-Rad Quantity One Software v4.6.2, USA).


## 2.11. Statistical Analysis

Statistical comparisons including unpairedt-test, one-way analysis of variance, and repeated measurement analysis of variance and figure creation were carried out by GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA), with P<0.05 showing statistical significance. All data were summarized by mean ± standard deviation.


## 3. Results

### 3.1. Inhibition of miR-29b-1-5p Ameliorated LPS-Induced MH-S Injury in vitro

An enhanced miR-29b-1-5p expression was observed in MH-S cells after LPS induction (Figure1(a)). We next manipulated miR-29b-1-5p in LPS-conditioned MH-S cells with miR-29b-1-5p inhibitor (Figure 1(b)) to confirm the functional role of miR-29b-1-5p in the development of ALI. ELISA was performed to measure the production of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) in the supernatant derived from LPS-conditioned MH-S cells (Figure 1(c)), and the results showed that the levels of pro-inflammatory cytokines were increased in the supernatant derived from LPS-conditioned MH-S cells. Inhibited miR-29b-1-5p by its specific inhibitor reduced the release of pro-inflammatory cytokines in the supernatant derived from LPS-conditioned MH-S cells. As shown by data obtained from cell viability and apoptosis assays, inhibited miR-29b-1-5p by its specific inhibitor protected MH-S cells against LPS-induced injury as evidenced by enhanced viability and inhibited apoptosis (Figures 1(d) and 1(e)). Overall, we concluded that the inhibition of miR-29b-1-5p protected MH-S cells against LPS-induced injury and inflammatory response in vitro.Figure 1
Inhibition of miR-29b-1-5p protected MH-S cells against LPS-induced injury and inflammatory responsein vitro. (a) The expression of miR-29b-1-5p in untreated MH-S cells and LPS-conditioned MH-S cells was determined using qRT-PCR. (b) The inhibition of miR-29b-1-5p in LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor was confirmed using qRT-PCR. (c) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant derived from LPS-conditioned MH-S cells were measured using the ELISA method. (d) The viability of LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor was evaluated using the CCK-8 assay. (e) The apoptosis rate of LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor. ∗, p<0.05 vs. MH-S cells and #p<0.05 vs. LPS-conditioned MH-S cells.
(a)(b)(c)(d)(e)


### 3.2. miR-29b-1-5p Regulated LPS-Induced MH-S Injury In Vitro through the Akt/ERK Signaling Pathway via RTN4

We next decipher the underlying mechanism by which miR-29b-1-5p modulates the development of ALI. We performed a miRNA-mRNA prediction across the miRWalk and RNA22 databases (Figure2(a)). We speculate that RTN4 may be implicated in the regulation mechanism of miR-29b-1-5p in ALI. Initially, dual-luciferase reporter gene assay revealed that the luciferase activity of RTN4-Wt was declined after adding exogenous miR-29b-1-5p, while we found an enhanced luciferase activity of RTN4-Wt after miR-29b-1-5p inhibitor transfection (Figure 2(b)). The inhibition of RTN4-B could suppress the activation of Akt in proliferative diabetic retinopathy [17], while the activation of the Akt signaling pathway attenuates LPS-induced inflammatory in lungs [18]. Thus, we attempt to examine the expression pattern of RTN4 and the regulation of miR-29b-1-5p on the RTN4 and the Akt/ERK signaling pathway in the context of ALI. The findings (Figure 2(c)) displayed that the mRNA level of RTN4 and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, was declined in LPS-conditioned MH-S cells following LPS induction. We observed that partial loss of miR-29b-1-5p function by its specific inhibitor enhanced the mRNA level of RTN4 mRNA and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, in LPS-conditioned MH-S cells. The result was also confirmed by the gain-of-function study using miR-29b-1-5p mimic. Accordingly, we delivered RTN4 expression vector into MH-S cells to achieve RTN4-overexpressed MH-S cells. Expression vector containing the RTN4 gene mimicked the effect of miR-29b-1-5p inhibitor on the Akt/ERK signaling pathway and enhanced the mRNA level of RTN4 mRNA and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, in LPS-conditioned MH-S cells. Besides, we determined lower expression of RTN4 in LPS-conditioned MH-S cells treated with miR-29b-1-5p mimic and RTN4 expression vector in combination than in LPS-conditioned MH-S cells treated with RTN4 expression vector alone, suggesting miR-29b-1-5p negatively regulated the RTN4. To demonstrate that RTN4 is indeed responsible for the regulation of miR-29b-1-5p on LPS-induced MH-S injury, we performed the ELISA method (Figure 2(d)), CCK-8 assay (Figure 2(e)), and Annexin V-FITC/PI-labeled flow cytometric analysis (Figure 2(f)) in LPS-conditioned MH-S cells treated with RTN4 expression vector and/or miR-29b-1-5p mimic. Likewise, we found RTN4 expression vector exerted similar effects on the pro-inflammatory cytokine production, cell viability, and apoptosis in LPS-conditioned MH-S cells, as miR-29b-1-5p inhibitor. RTN4 overexpression attenuated the pro-inflammatory cytokine production in the supernatant derived from LPS-conditioned MH-S cells, enhanced MH-S cell viability, and inhibited the apoptosis, suggesting that RTN4 could protect MH-S cells against injury and inflammatory response caused by LPS in vitro. Furthermore, the RTN4 gene resisting to miR-29b-1-5p attack on MH-S cells and inflammatory response was also detected. The aforementioned results together demonstrate RTN4 plays anti-apoptotic and anti-inflammatory roles in LPS-induced ALI and is implicated in the regulation of miR-29b-1-5p in LPS-induced ALI.Figure 2
miR-29b-1-5p negatively regulates the RTN4 and Akt/ERK signaling pathway, thus promoting LPS-induced injury and inflammatory responsein vitro. (a) Analysis of the miRWalk and RNA22 databases. (b) Luciferase activity assays of the RTN4-Wt and RTN4-Mut in the presence of miR-29b-1-5p mimic or inhibitor. (c), The mRNA level of RTN4 was determined by using qRT-PCR and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent by using Western blot analysis. (d) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant derived from LPS-conditioned MH-S cells were measured using the ELISA method. (e) The viability of LPS-conditioned MH-S cells was evaluated using CCK-8 assay at indicated time points. (f) The apoptosis rate of LPS-conditioned MH-S cells. ∗, p<0.05 vs. MH-S cells, #p<0.05 vs. LPS-conditioned MH-S cells, and p<0.05 vs. MH-S cells treated with miR-29b-1-5p mimic.
(a)(b)(c)(d)(e)(f)


### 3.3. Upregulated miR-29b-1-5p in ALI Animal Model

Our next effort is to confirm the contributory role of miR-29b-1-5p and inhibitory role of RTN4 in ALIin vivo. The lungs of normal mice presented intact alveolar structure, with the absence of lymphocyte infiltration. However, we observed evidenced pathological changes in the lungs of LPS-conditioned mice, as shown by thickening alveolar wall, collapsed alveolar, and red blood cell and inflammatory cell infiltration. After LPS induction, the W/D ratio and MPO activity were increased in mice (Figures 3(a) and 3(b)). The ELISA method determined elevated levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) elevated in the BALF of LPS-conditioned mice (Figure 3(c)). As we expected, an increased miR-29b-1-5p expression with a declined RTN4 expression was found in the mouse lung tissue after LPS treatment (Figure 3(d)).Figure 3
miR-29b-1-5p is overexpressed in LPS-induced ALIin vivo. (a), The W/D ratio of normal mouse lung and LPS-induced ALI mouse lung. (b) The MPO activity of lung tissues in normal mice and LPS-induced ALI mice. (c) The levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) in the BALF of normal mice and LPS-induced ALI mice. (d) Detection of miR-29b-1-5p and RTN4 expressions in mouse lung tissues was determined using the qRT-PCR and Western blot analyses. ∗, p<0.05vs. normal mice.
(a)(b)(c)(d)


### 3.4. miR-29b-1-5p-Mediated Inflammatory Response and Lung Injury in ALI Animal Model through the Akt/ERK Signaling Pathway via RTN4

In this part, the LPS-induced ALI mouse model was injected with agomir and antagomir of miR-29b-1-5p and RTN4 expression vectorvia tail vein to perturb the expression of miR-29b-1-5p and RTN4 (Figure 4(a)). As we expected, the expression of RTN4 in lung tissues of LPS-induced ALI mice was declined and increased in response to injection with agomir and antagomir of miR-29b-1-5p. Accordingly, caudal vein injection of miR-29b-1-5p antagomir or RTN4 expression vector contributed to decreased W/D ratio, weakened MPO activity, and declined levels of pro-inflammatory cytokines in the BALF in LPS-conditioned mice (Figures 4(b)–4(d)). We found an increased ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in LPS-conditioned mice injected with miR-29b-1-5p antagomir or RTN4 expression vector (Figure 4(e)), while we observed a declined ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in LPS-conditioned mice injected with miR-29b-1-5p agomir. Meanwhile, when LPS-conditioned mice were injected with miR-29b-1-5p antagomir and RTN4 expression vector in combination, we observed the overexpression of RTN4 rescued LPS-conditioned mice against miR-29b-1-5p attack, as evidenced by decreased W/D ratio and MPO activity, activation of the Akt/ERK signaling pathway, and reduced release of pro-inflammatory cytokines. The aforementioned results together prove the hypothesis that miR-29b-1-5p mediates inflammatory response and lung injury in LPS-induced ALI in vivo through the Akt/ERK signaling pathway via the RTN4.Figure 4
miR-29b-1-5p mediates inflammatory response and lung injury in LPS-induced ALIin vivo through the Akt/ERK signaling pathway via the RTN4. (a) qRT-PCR and Western blot analyses of miR-29b-1-5p and RTN4 expressions in mouse lung tissues of LPS-conditioned mice. (b) W/D ratio of LPS-induced mouse lungs. (c) MPO activity of LPS-induced mouse lungs. (d) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the BALF of normal mice and LPS-induced ALI mice. (e) The ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in lung tissues of LPS-conditioned mice. ∗, p<0.05vs. LPS-induced ALI mice and #p<0.05vs. miR-29b-1-5p agomir.
(a)(b)(c)(d)(e)miR-29b-1-5p mediated pulmonary interstitial fibrosis in ALI animal model by targeting RTN4.Subsequently, we determined the expression of TGF-β1, E-cadherin, α-SMA, and vimentin by using qRT-PCR and Western blot analyses to further confirm the occurrence of EMT (Figures 5(a) and 5(b)). The results revealed that TGF-β1, α-SMA, and vimentin mRNA and protein expressions declined but the E-cadherin was increased in LPS-conditioned mice injected with miR-29b-1-5p antagomir or RTN4 expression vector, while the results were opposite in those injected with agomir. Likewise, the overexpression of RTN4 by its expression vector mimicked the effect of miR-29b-1-5p inhibition on the expression of TGF-β1, E-cadherin, α-SMA, and vimentin in the ALI animal model.Figure 5
miR-29b-1-5p mediated pulmonary interstitial fibrosis in the ALI animal model by targeting RTN4. LPS-induced ALI mice were injected with miR-29b-1-5p agomir, miR-29b-1-5p antagomir, miR-29b-1-5p NC, RTN4 expression vector, and empty vector alone or in combination as requiredvia caudal vein to manipulate the expression of miR-29b-1-5p and RTN4 in vivo. (a) The mRNA levels of TGF-β1, E-cadherin, α-SMA, and vimentin in LPS-induced ALI mouse lung tissues were determined using qRT-PCR. (b) The protein expressions of TGF-β1, E-cadherin, α-SMA, and vimentin in LPS-induced ALI mouse lung tissues were determined using Western blot analysis. ∗, p<0.05 vs. LPS-induced ALI mice and #p<0.05 vs. miR-29b-1-5p agomir.
(a)(b)


## 3.1. Inhibition of miR-29b-1-5p Ameliorated LPS-Induced MH-S Injury in vitro

An enhanced miR-29b-1-5p expression was observed in MH-S cells after LPS induction (Figure1(a)). We next manipulated miR-29b-1-5p in LPS-conditioned MH-S cells with miR-29b-1-5p inhibitor (Figure 1(b)) to confirm the functional role of miR-29b-1-5p in the development of ALI. ELISA was performed to measure the production of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) in the supernatant derived from LPS-conditioned MH-S cells (Figure 1(c)), and the results showed that the levels of pro-inflammatory cytokines were increased in the supernatant derived from LPS-conditioned MH-S cells. Inhibited miR-29b-1-5p by its specific inhibitor reduced the release of pro-inflammatory cytokines in the supernatant derived from LPS-conditioned MH-S cells. As shown by data obtained from cell viability and apoptosis assays, inhibited miR-29b-1-5p by its specific inhibitor protected MH-S cells against LPS-induced injury as evidenced by enhanced viability and inhibited apoptosis (Figures 1(d) and 1(e)). Overall, we concluded that the inhibition of miR-29b-1-5p protected MH-S cells against LPS-induced injury and inflammatory response in vitro.Figure 1
Inhibition of miR-29b-1-5p protected MH-S cells against LPS-induced injury and inflammatory responsein vitro. (a) The expression of miR-29b-1-5p in untreated MH-S cells and LPS-conditioned MH-S cells was determined using qRT-PCR. (b) The inhibition of miR-29b-1-5p in LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor was confirmed using qRT-PCR. (c) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant derived from LPS-conditioned MH-S cells were measured using the ELISA method. (d) The viability of LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor was evaluated using the CCK-8 assay. (e) The apoptosis rate of LPS-conditioned MH-S cells following the transfection of miR-29b-1-5p inhibitor. ∗, p<0.05 vs. MH-S cells and #p<0.05 vs. LPS-conditioned MH-S cells.
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## 3.2. miR-29b-1-5p Regulated LPS-Induced MH-S Injury In Vitro through the Akt/ERK Signaling Pathway via RTN4

We next decipher the underlying mechanism by which miR-29b-1-5p modulates the development of ALI. We performed a miRNA-mRNA prediction across the miRWalk and RNA22 databases (Figure2(a)). We speculate that RTN4 may be implicated in the regulation mechanism of miR-29b-1-5p in ALI. Initially, dual-luciferase reporter gene assay revealed that the luciferase activity of RTN4-Wt was declined after adding exogenous miR-29b-1-5p, while we found an enhanced luciferase activity of RTN4-Wt after miR-29b-1-5p inhibitor transfection (Figure 2(b)). The inhibition of RTN4-B could suppress the activation of Akt in proliferative diabetic retinopathy [17], while the activation of the Akt signaling pathway attenuates LPS-induced inflammatory in lungs [18]. Thus, we attempt to examine the expression pattern of RTN4 and the regulation of miR-29b-1-5p on the RTN4 and the Akt/ERK signaling pathway in the context of ALI. The findings (Figure 2(c)) displayed that the mRNA level of RTN4 and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, was declined in LPS-conditioned MH-S cells following LPS induction. We observed that partial loss of miR-29b-1-5p function by its specific inhibitor enhanced the mRNA level of RTN4 mRNA and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, in LPS-conditioned MH-S cells. The result was also confirmed by the gain-of-function study using miR-29b-1-5p mimic. Accordingly, we delivered RTN4 expression vector into MH-S cells to achieve RTN4-overexpressed MH-S cells. Expression vector containing the RTN4 gene mimicked the effect of miR-29b-1-5p inhibitor on the Akt/ERK signaling pathway and enhanced the mRNA level of RTN4 mRNA and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent, in LPS-conditioned MH-S cells. Besides, we determined lower expression of RTN4 in LPS-conditioned MH-S cells treated with miR-29b-1-5p mimic and RTN4 expression vector in combination than in LPS-conditioned MH-S cells treated with RTN4 expression vector alone, suggesting miR-29b-1-5p negatively regulated the RTN4. To demonstrate that RTN4 is indeed responsible for the regulation of miR-29b-1-5p on LPS-induced MH-S injury, we performed the ELISA method (Figure 2(d)), CCK-8 assay (Figure 2(e)), and Annexin V-FITC/PI-labeled flow cytometric analysis (Figure 2(f)) in LPS-conditioned MH-S cells treated with RTN4 expression vector and/or miR-29b-1-5p mimic. Likewise, we found RTN4 expression vector exerted similar effects on the pro-inflammatory cytokine production, cell viability, and apoptosis in LPS-conditioned MH-S cells, as miR-29b-1-5p inhibitor. RTN4 overexpression attenuated the pro-inflammatory cytokine production in the supernatant derived from LPS-conditioned MH-S cells, enhanced MH-S cell viability, and inhibited the apoptosis, suggesting that RTN4 could protect MH-S cells against injury and inflammatory response caused by LPS in vitro. Furthermore, the RTN4 gene resisting to miR-29b-1-5p attack on MH-S cells and inflammatory response was also detected. The aforementioned results together demonstrate RTN4 plays anti-apoptotic and anti-inflammatory roles in LPS-induced ALI and is implicated in the regulation of miR-29b-1-5p in LPS-induced ALI.Figure 2
miR-29b-1-5p negatively regulates the RTN4 and Akt/ERK signaling pathway, thus promoting LPS-induced injury and inflammatory responsein vitro. (a) Analysis of the miRWalk and RNA22 databases. (b) Luciferase activity assays of the RTN4-Wt and RTN4-Mut in the presence of miR-29b-1-5p mimic or inhibitor. (c), The mRNA level of RTN4 was determined by using qRT-PCR and the protein expression of RTN4A/B, the p-Akt and p-ERK1/2 extent by using Western blot analysis. (d) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the supernatant derived from LPS-conditioned MH-S cells were measured using the ELISA method. (e) The viability of LPS-conditioned MH-S cells was evaluated using CCK-8 assay at indicated time points. (f) The apoptosis rate of LPS-conditioned MH-S cells. ∗, p<0.05 vs. MH-S cells, #p<0.05 vs. LPS-conditioned MH-S cells, and p<0.05 vs. MH-S cells treated with miR-29b-1-5p mimic.
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## 3.3. Upregulated miR-29b-1-5p in ALI Animal Model

Our next effort is to confirm the contributory role of miR-29b-1-5p and inhibitory role of RTN4 in ALIin vivo. The lungs of normal mice presented intact alveolar structure, with the absence of lymphocyte infiltration. However, we observed evidenced pathological changes in the lungs of LPS-conditioned mice, as shown by thickening alveolar wall, collapsed alveolar, and red blood cell and inflammatory cell infiltration. After LPS induction, the W/D ratio and MPO activity were increased in mice (Figures 3(a) and 3(b)). The ELISA method determined elevated levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) elevated in the BALF of LPS-conditioned mice (Figure 3(c)). As we expected, an increased miR-29b-1-5p expression with a declined RTN4 expression was found in the mouse lung tissue after LPS treatment (Figure 3(d)).Figure 3
miR-29b-1-5p is overexpressed in LPS-induced ALIin vivo. (a), The W/D ratio of normal mouse lung and LPS-induced ALI mouse lung. (b) The MPO activity of lung tissues in normal mice and LPS-induced ALI mice. (c) The levels of pro-inflammatory cytokines (IL-1β, IL-8, TNF-α, and IL-6) in the BALF of normal mice and LPS-induced ALI mice. (d) Detection of miR-29b-1-5p and RTN4 expressions in mouse lung tissues was determined using the qRT-PCR and Western blot analyses. ∗, p<0.05vs. normal mice.
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## 3.4. miR-29b-1-5p-Mediated Inflammatory Response and Lung Injury in ALI Animal Model through the Akt/ERK Signaling Pathway via RTN4

In this part, the LPS-induced ALI mouse model was injected with agomir and antagomir of miR-29b-1-5p and RTN4 expression vectorvia tail vein to perturb the expression of miR-29b-1-5p and RTN4 (Figure 4(a)). As we expected, the expression of RTN4 in lung tissues of LPS-induced ALI mice was declined and increased in response to injection with agomir and antagomir of miR-29b-1-5p. Accordingly, caudal vein injection of miR-29b-1-5p antagomir or RTN4 expression vector contributed to decreased W/D ratio, weakened MPO activity, and declined levels of pro-inflammatory cytokines in the BALF in LPS-conditioned mice (Figures 4(b)–4(d)). We found an increased ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in LPS-conditioned mice injected with miR-29b-1-5p antagomir or RTN4 expression vector (Figure 4(e)), while we observed a declined ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in LPS-conditioned mice injected with miR-29b-1-5p agomir. Meanwhile, when LPS-conditioned mice were injected with miR-29b-1-5p antagomir and RTN4 expression vector in combination, we observed the overexpression of RTN4 rescued LPS-conditioned mice against miR-29b-1-5p attack, as evidenced by decreased W/D ratio and MPO activity, activation of the Akt/ERK signaling pathway, and reduced release of pro-inflammatory cytokines. The aforementioned results together prove the hypothesis that miR-29b-1-5p mediates inflammatory response and lung injury in LPS-induced ALI in vivo through the Akt/ERK signaling pathway via the RTN4.Figure 4
miR-29b-1-5p mediates inflammatory response and lung injury in LPS-induced ALIin vivo through the Akt/ERK signaling pathway via the RTN4. (a) qRT-PCR and Western blot analyses of miR-29b-1-5p and RTN4 expressions in mouse lung tissues of LPS-conditioned mice. (b) W/D ratio of LPS-induced mouse lungs. (c) MPO activity of LPS-induced mouse lungs. (d) The levels of IL-1β, IL-8, TNF-α, and IL-6 in the BALF of normal mice and LPS-induced ALI mice. (e) The ratio of p-Akt/total Akt and p-ERK1/2/total ERK1/2 in lung tissues of LPS-conditioned mice. ∗, p<0.05vs. LPS-induced ALI mice and #p<0.05vs. miR-29b-1-5p agomir.
(a)(b)(c)(d)(e)miR-29b-1-5p mediated pulmonary interstitial fibrosis in ALI animal model by targeting RTN4.Subsequently, we determined the expression of TGF-β1, E-cadherin, α-SMA, and vimentin by using qRT-PCR and Western blot analyses to further confirm the occurrence of EMT (Figures 5(a) and 5(b)). The results revealed that TGF-β1, α-SMA, and vimentin mRNA and protein expressions declined but the E-cadherin was increased in LPS-conditioned mice injected with miR-29b-1-5p antagomir or RTN4 expression vector, while the results were opposite in those injected with agomir. Likewise, the overexpression of RTN4 by its expression vector mimicked the effect of miR-29b-1-5p inhibition on the expression of TGF-β1, E-cadherin, α-SMA, and vimentin in the ALI animal model.Figure 5
miR-29b-1-5p mediated pulmonary interstitial fibrosis in the ALI animal model by targeting RTN4. LPS-induced ALI mice were injected with miR-29b-1-5p agomir, miR-29b-1-5p antagomir, miR-29b-1-5p NC, RTN4 expression vector, and empty vector alone or in combination as requiredvia caudal vein to manipulate the expression of miR-29b-1-5p and RTN4 in vivo. (a) The mRNA levels of TGF-β1, E-cadherin, α-SMA, and vimentin in LPS-induced ALI mouse lung tissues were determined using qRT-PCR. (b) The protein expressions of TGF-β1, E-cadherin, α-SMA, and vimentin in LPS-induced ALI mouse lung tissues were determined using Western blot analysis. ∗, p<0.05 vs. LPS-induced ALI mice and #p<0.05 vs. miR-29b-1-5p agomir.
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## 4. Discussion

Despite tremendous efforts in clinical trials, ALI still brings tremendous fatality and morbidity rates [19]. Recently, the functional roles of miRNAs focusing on ALI pathogenesis have been well characterized [9]. Herein, we attempt to elucidate the effect of miR-29b-1-5p on inflammatory response and pulmonary fibrosis in LPS-conditioned ALI. The study reveals that the inhibition of miR-29b could relieve inflammatory response and pulmonary fibrosis in LPS-conditioned ALI by modulating RTN4 and the Akt/ERK pathway.In this report, our results demonstrated an elevated miR-29b-1-5p in LPS-conditioned MH-S cells and in LPS-conditioned ALI mice. As reported previously by Zhang et al., inhibited miR-29b could reduce the numbers of apoptotic cells and inflammatory reaction in H9c2 cells following LPS treatment [20], suggesting the upregulation of miR-29b when exposure to LPS. Likewise, we determined miR-29b-1-5p in LPS-induced murine alveolar macrophages MH-S and LPS-induced murine models. LPS is still a common stimulator used to induce ALI in vivo and in vitro. Therefore, we may conclude the overexpression of miR-29b-1-5p following ALI. In addition to Zhang et al., Long et al. reported upregulated miR-29b-1-5p in hearts following I/R injury and in hydrogen peroxide-treated cardiomyocytes [13], suggesting that miR-29b-1-5p appears to upregulate following tissue injury. A large set of functional miRNAs have been well characterized, which can regulate gene expression at various levels, including transcription and post-transcriptional processing. Furthermore, we determined an upregulation of RTN4 expression in response to miR-29b-1-5p inhibition. In addition to that, we found RTN4 overexpression resisting to miR-29a-1-5p overexpression in LPS-induced murine alveolar macrophages MH-S and LPS-induced murine models both, suggesting miR-29a-1-5p-mediated ALI partially by targeting RTN4. Till now, the demonstration that miR-29a-1-5p-mediated ALI partially by targeting RTN4 has not been reported in ALI. The family of RTN4 encompasses three different isoforms, among which RTN4-B is highly expressed in the lung tissue. A murine study demonstrated RTN4-B overexpression ameliorates lung injury, alveolar protein exudation, and neutrophil infiltration in lipopolysaccharide (LPS)-induced ALI mice, suggesting protective effects of RTN4-B against ALI [16]. Likewise, the loss of RTN4-B was identified as an unfavorable feature in the context of intrahepatic cholangiocarcinoma [21]. Therefore, miR-29b-1-5p-mediated RTN4 inhibition may explain the development of ALI.Additionally, the data in the present study support the notion that miR-29b-1-5p regulates RTN4 expression and its overexpression contributes to the release of pro-inflammatory cytokines, IL-1β, IL-8, TNF-α, and IL-6. In addition to inflammation, miR-29b-1-5p upregulation following LPS stimulation was associated with cell apoptosis in ALI. Exposure to LPS often leads to the accumulation of inflammatory cells in alveolus as well as inflammatory cytokine secretion [22]. miR-29b-1-5p upregulation was found to increase endothelial permeability and apoptosis, and increase the expression of NF-κB and cell adhesion molecule-1 in atherosclerosis [23]. A disruption of the alveolar epithelial barrier and an enhanced capillary endothelial permeability commonly accompany with ALI [24]. Furthermore, LPS stimulation was followed by an elevated expression of vascular cell adhesion molecule-1 [25]. Therefore, it is reasonable that miR-29b-1-5p upregulation following LPS stimulation triggers the inflammation and apoptosis in LPS-induced ALI. Interestingly, we found RTN4 overexpression negated the inducible role of miR-29b-1-5p in inflammation during ALI. The products of RTN4 gene may be multifunctional, modulating the apoptosis, inflammation, tumor development, and neuronal regeneration. In a previous murine study, mice with Th2-driven lung inflammation exhibited a loss of Nogo expression in the airway epithelium and smooth muscle when compared to nonallergic mice [26]. RTN4-B required for tissue repair was also observed from previous evidence [27]. Additionally, our study demonstrated miR-29b-1-5p mediated pulmonary interstitial fibrosis in ALI via RTN4. In this study, TGF-β1, E-cadherin, α-SMA, and vimentin were determined to reflect the degree of fibrosis in mouse lungs following LPS stimulation. As evidenced by our experimental ALI mouse models, the expression of TGF-β1, α-SMA, and vimentin was declined but the E-cadherin was increased in LPS-conditioned mice with miR-29b-1-5p inhibition or with RTN4 expression. Similar to our study, RTN4B is nonparenchymal cells in the liver and its expression was downregulated with the progression of liver fibrosis [21].In conclusion, the findings obtained in this study provide evidence that the inhibition of miR-29b-1-5p could decelerate the inflammation, apoptosis, and pulmonary interstitial fibrosis in LPS-induced ALI. Likewise, we demonstrated the contributory role of miR-29b-1-5p overexpression in ALI was achieved by functioning as a negative regulator of RTN4. In addition to that, the PI3K/AKT pathway was found to be suppressed in the presence of miR-29b-1-5p or to be activated in the presence of RTN4 in LPS-induced ALI, while a further demonstration that this signaling pathway engages in the regulation of miR-29b and RTN4 in ALI is required. Although the present study shows preliminary nature, clinical translation can be improved by the drug delivery system targeting miR-29b-1-5p or restoring RTN4 for ALI.



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