The aim of the present study was to investigate the regulatory functions of microRNA (miR)-26a-5p on lipopolysaccharide (LPS)-induced acute lung injury (ALI) and its molecular mechanisms. The role of miR-26a-5p on an ALI mouse model was evaluated by examining the histological changes, wet/dry (W/D) ratio, myeloperoxidase (MPO) activity, malondialdehyde (MDA) expression levels in lung tissues and the survival of ALI mice. Moreover, the protein concentration and the number of neutrophils and lymphocytes in bronchoalveolar lavage fluid (BALF) was analyzed. To explore the effect of miR-26a-5p on inflammatory responses and apoptosis, the expression levels of tumour necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 and apoptosis were measured by ELISA, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling staining and flow cytometry in BALF, A549 cells and lung tissues. B-cell lymphoma-2 (Bcl-2), Bax and cleaved caspase-3 in lung tissues were measured by western blotting and reverse transcription-quantitative PCR. Connective tissue growth factor (CTGF) was predicted as a direct target of miR-26a-5p using dual luciferase reporter assay. The present study sought to determine whether CTGF overexpression reversed the effect of miR-26a-5p on apoptosis and inflammatory responses in LPS-induced A549 cells. The data revealed that miR-26a-5p overexpression ameliorated LPS-induced ALI, which was implicated by fewer histopathological changes, W/D ratio, apoptosis in lung tissues and the survival of ALI mice. Moreover, miR-26a-5p overexpression alleviated LPS-induced inflammatory responses in ALI mice via the reduction of total protein, neutrophil and lymphocyte counts and the expression levels of TNF-α, IL-1β, IL-6, MDA and MPO activity in BALF. Similarly, miR-26a-5p overexpression decreased apoptosis and the expression of TNF-α, IL-1β and IL-6 in LPS-induced A549 cells. CTGF was a direct target of miR-26a-5p. CTGF overexpression reversed the effect of miR-26a-5p on cell apoptosis and inflammatory responses in LPS-induced A549 cells. The present study demonstrated that miR-26a-5p could attenuate lung inflammation and apoptosis in LPS-induced ALI by targeting CTGF.
Acute lung injury (ALI) is characterized by serious pulmonary inflammatory responses with a high incidence of morbidity and mortality (
MicroRNAs (miRNAs; miRs) are a class of short non-protein coding RNAs with lengths of 19–25 nucleotides, which modulate target gene expression at the posttranscriptional level (
Connective tissue growth factor (CTGF) is a multifunctional matricellular protein that belongs to the CTGF-Cyr61/Cef10-Nov (CCN) family. It was first reported in 1991 by Bradham
The present research aimed to identify the potential effects of miRNA-26a-5p on LPS-induced ALI and its related molecular mechanisms
A total of 230 male C57BL/6 mice (6–8 weeks; 20–25 g) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in climate-controlled quarters with a 12-h light/dark cycle and provided water and standard laboratory chow
The mice were intratracheally injected with 5 mg/kg LPS (Beijing Solabio Science & Technology C., Ltd.), dissolved in phosphate buffer saline (PBS). A total of 24 h prior to LPS treatment, the mice were administered with intraperitoneal injections of miR-26a-5p mimic (20 mg/kg) or miR-26a-5p mimic negative control (NC) (20 mg/kg) twice per day for 2 days consecutively. Subsequently, the mice were randomly divided into five groups (n=38/group): Control group (normal mice without any treatment), Sham group (received PBS), LPS group (received LPS), LPS + mimic negative control (NC) group (received LPS and miR-26a-5p mimic NC) and LPS + miR-26a-5p mimic group (received LPS and miR-26a-5p mimic). All mice were sacrificed by cervical dislocation under anesthesia via intraperitoneal injection of 50 mg/kg pentobarbital sodium at 24 h after LPS treatment for the subsequent experiment. The criteria used to confirm death were as follows: Cessation of heartbeat for more than 5 min and no pupillary reflex to strong light. The miR-26a-5p mimic and miR-26a-5p mimic NC were purchased from Takara Biotechnology Co., Ltd. The sequences used were as follows: miR-26a-5p mimic, 5′-UUCAAGUAAUCCAGGAUAGGCU-3′ (sense); 5′-CCUAUCCUGGAUUACUUGAAUU-3′ (antisense); and miR-26a-5p mimic NC, 5′-UUCUCCGAACGUGUCACGUTT-3′ (sense); 5′-ACGUGACACGUUCGGAGAATT-3′ (antisense).
In addition, survival experiments were performed in the four mice groups (n=10/group; Sham, LPS, LPS + mimic NC, LPS + miR-26a-5p mimic). The survival of mice was monitored for 84 h.
Lung edema was evaluated according to the wet/dry weight ratio of lung tissues. After the mice (n=6) were sacrificed, the right lungs were excised and immediately weighed to obtain the wet weight. Subsequently, the lung samples were dried at 80°C for 48 h to obtain the dry weight (Lung wet/dry ratio=wet weight/dry weight).
After the mice (n=6) were sacrificed, the right lung tissues were collected and homogenized in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid containing 0.5% cetyltrimethyl ammonium bromide. Then, the activity of MPO (cat. no. A044) and the content of MDA (cat. no. A003-1) were measured by using the corresponding test kits obtained from Nanjing Jiancheng Bioengineering Institute in accordance with the manufacturer's instructions.
After the mice (n=6) were sacrificed, the BALF was collected by injection and retraction of 1.5 ml PBS three times, and was centrifuged at 1,200 × g for 10 min at 4°C. Subsequently, the supernatants were collected for the assessment of total protein concentration by the bicinchoninic acid (BCA) method. The cell pellet was resuspended in PBS, and the neutrophil and lymphocyte counts were determined using a hemocytometer following Wright-Giemsa staining for 10 min at room temperature.
After the mice (n=5) were sacrificed, the right lung tissues were collected and fixed in 4% formaldehyde at room temperature for 24 h. Then, the lung tissues were dehydrated in graded concentrations of ethanol, embedded in paraffin, and transversely cut into 5-µm thick sections. Finally, the histological changes in lung tissues were analyzed by H&E staining under an optical microscope (magnification, ×100). In addition, the histological score of the lung tissues was calculated by assessing the alveolar congestion, hemorrhage, inflammatory cell infiltration and alveolar wall thickness, and graded according to a five-point scale from 0 to 4 as follows: 0=no damage, l=mild damage, 2=moderate damage, 3=severe damage and 4=very severe damage (
After the mice (n=5) were sacrificed, the apoptotic cells in the harvested lung tissues were detected by using the TUNEL staining kit supplied by Shanghai Ruisai Biotechnology Co., Ltd. following the manufacturer's protocol. Five random fields were viewed in each section and the rate of positive cells was analyzed under a fluorescence microscope at a magnification of ×400.
The human type II alveolar epithelial cells (A549), supplied by American Type Culture Collection, were maintained in Dulbecco's Modified Eagle's Medium (DMEM; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) and 1% penicillin/streptomycin (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO2. The miR-26a-5p mimic, pcDNA3.1-CTGF and their negative controls were obtained from Takara Biotechnology Co., Ltd. Briefly, 10 nM miR-26a-5p mimic or pcDNA3.1-CTGF were transfected into A549 cells (6×106) using Lipofectamine® 3000 (Invitrogen; Thermo Fisher Scientific, Inc.), at room temperature for 48 h according to the manufacturer's protocol. The transfected A549 cells were randomly divided into the Control group (no treatment), LPS group (treated with LPS), LPS + mimic NC group (treated with LPS and miR-26a-5p mimic negative control), LPS + miR-26a-5p mimic group (treated with LPS and miR-26a-5p mimic), LPS + miR-26a-5p mimic + oe-CTGF NC group (treated with LPS, miR-26a-5p mimic and pcDNA3.1-CTGF NC) and LPS + miR-26a-5p mimic + oe-CTGF (treated with LPS, miR-26a-5p mimic and pcDNA3.1-CTGF). After transfection for 48 h, the A549 cells were treated with LPS (Sigma-Aldrich; Merck KGaA) for 24 h and analyzed.
To assess cell viability, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used. The transfected A549 cells were seeded into 96-well plates at a density of 1×103 cells/well. After 24 h of LPS stimulation, A549 cells were incubated with 20 µl of MTT at a final concentration of 5 mg/ml (Sigma-Aldrich; Merck KGaA) in the dark for 4 h. Subsequently, dimethyl sulfoxide (DMSO, 150 µl) was added to each well. Finally, the cell viability was determined by measuring absorbances at 490 nm by using a microplate reader.
The A549 cells undergoing apoptosis were detected with an Annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Biosciences) following the manufacturer's instructions. Briefly, 24 h after LPS stimulation, the A549 cells from different groups were harvested, washed with PBS and resuspended in Annexin-binding buffer. Propidium iodide (PI) and FITC-conjugated Annexin V were added into the cell suspensions and maintained in the dark for 15 min at 37°C. The apoptotic cells were then analyzed using a flow cytometer (BD FACScalibur; BD Biosciences).
The levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α in mice BALF (n=6) and A549 cells were determined using an ELISA kit (BioLegend, Inc.) in accordance with the manufacturer's instructions. The ELISA kits for mice BALF were as follows: IL-1β (cat. no. 432601), IL-6 (cat. no. 431304) and TNF-α (cat. no. 430904). The ELISA kits for A549 cells were as follows: IL-1β (cat. no. 579409), IL-6 (cat. no. 430504) and TNF-α (cat. no. 430204).
TargetScan (
Lung tissue was collected after the mice (n=5) were sacrificed. Total RNA was extracted from A549 cells and lung tissues by TRIzol® Reagent (Thermo Fisher Scientific, Inc.), based on the manufacturer's protocols. Total RNA (2 µg) was added to the reverse transcription reaction to synthesize complementary DNA (cDNA) using SuperScript™ IV First-Strand Synthesis System (Invitrogen; Thermo Fisher Scientific, Inc.). Thereafter, RT-qPCR was performed on a 7500 Real-time PCR System with MirVana™ qRT-PCR miRNA (Invitrogen; Thermo Fisher Scientific, Inc.). The PCR reaction conditions were as follows: 95°C for 10 min followed by 40 cycles at 95°C for 10 sec, 60°C for 20 sec, and 72°C for 30 sec. The primers used in the present study were as follows: miR-26a-5p forward (F), 5′-GGATCCGCAGAAACTCCAGAGA-3′ and reverse (R), 5′-TTGGAGGAAAGACGATTTCCGT-3′; U6 F, 5′-GCGCGTCGTGTAAAGCGTTC-3′ and R, 5′-GTGCAGGGTCCGAGGT-3′; CTGF F, 5′-CAAGGGCCTCTTCTGTGACT-3′ and R, 5′-ACGTGCACTGGTACTTGCAG-3′; B-cell lymphoma-2 (Bcl-2) F, 5′-ATGTGTGTGGAGAGCGTCAA-3′ and R, 5′-GCCGGTTCAGGTACTCAGTC-3′; Bax F: 5′-TCTGACGGCAACTTCAACTG-3′, R: 5′-GGAGGAAGTCCAATGTCCAG-3′; cleaved caspase-3 F: 5′-CTCGGTCTGGTACAGATGTCG-3′, R: 5′-TGGCTCAGAAGCACACAAAC-3′; GAPDH F, 5′-GCACCGTCAAGGCTGAGAAC-3′ and R, 5′-ATGGTGGTGAAGACGCCAGT-3′. For the normalization of the expression levels, U6 and GAPDH were used as internal controls. The relative expression levels were calculated using the 2−ΔΔCq method (
Lung tissue was collected after the mice (n=5) were sacrificed. Total proteins were extracted from A549 cells and lung tissues by using lysis buffer (Beijing Solarbio Science & Technology Co., Ltd.) containing phenylmethylsulfonyl fluoride (PMSF). The concentration of proteins was measured with a BCA protein assay kit (Beyotime Institute of Biotechnology). Protein samples (50 µg) were loaded on 10% sodium dodecyl sulfate-polyacrylamide gel for electrophoresis and then transferred onto polyvinylidene difluoride (PVDF) membranes (Merck KGaA). After blocking with 5% skimmed milk for 2 h at room temperature, the membranes were incubated at 4°C overnight with the following primary antibodies supplied by Cell Signaling, Inc.: Bax (1:1,000; product no. 14796), cleaved caspase-3 (1:1,000; product no. 9661), Bcl-2 (1:1,000; product no. 3498), CTGF (1:1,000; product no. 86641) and GAPDH (1:1,000; product no. 5174). After washing with TBST (20% Tween-20) three times, the membranes were incubated with the horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:5,000; cat. no. 7040; Cell Signaling Technology, Inc.) at room temperature for 1 h. Finally, the protein bands were visualized with an ECL system (Thermo Fisher Scientific, Inc.) and then analyzed by using Image Lab™ Software (version 3.0; Bio-Rad Laboratories, Inc.).
The statistical analysis was performed using SPSS 23.0 software (IBM Corp.). Data in the present study are presented as the mean ± standard deviation (SD). Statistical differences were analyzed using the unpaired Student's t-test or one-way analysis of variance with Tukey's multiple comparison post-hoc test. Survival rates were assessed by the Kaplan-Meier method, and survival curves were compared by log-rank tests. P<0.05 was considered to indicate a statistically significant difference. All experimental data were obtained from at least three independent experiments.
The results of RT-qPCR revealed that the expression of miR-26a-5p was significantly decreased in the LPS group compared with the Sham group (P<0.01;
The protein expression levels and the number of neutrophils and lymphocytes in BALF were significantly increased in the LPS group compared with the Sham group (P<0.01;
To evaluate the cell apoptosis in lung tissues, TUNEL staining was performed (
As revealed in
TargetScan predicted that the binding site of miR-26a-5p was the 3′-UTR region in CTGF (
As revealed in
ALI is a serious lung disease that is often accompanied by acute inflammatory responses and can lead to respiratory failure (
Recent studies have reported that miRNAs, as gene expression switches, have an important role in the progression of ALI (
Previous studies have indicated that ALI is featured with the production of inflammatory factors, inflammatory cell infiltration and pulmonary epithelial cell apoptosis (
CTGF, also referred to as CCN family protein 2 (CCN2) and one of six members of cysteine-rich, secreted, heparin-binding proteins with a modular structure, has an important role in tissue remodeling and fibrosis (
In summary, the present study confirmed that the expression of miR-26a-5p was downregulated in LPS-induced ALI mice and in A549 cells, while CTGF expression was upregulated. In addition, miR-26a-5p was demonstrated to attenuate lung inflammation and apoptosis in LPS-induced ALI by targeting CTGF. The findings of the present study provide new perspectives on miRNA-based diagnostic approaches against LPS-induced ALI. A key limitation of the present study was that only the effect of miR-26a-5p on CTGF expression was assessed; therefore, further investigation is required to verify the protective effect of miR-26a-5p against LPS-induced ALI.
Not applicable.
No funding was received.
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.
ZF designed and conceived the study. HL and TY performed the research, analyzed data and wrote the manuscript. All authors have read and approved the final version of the manuscript.
The protocol of this research has been approved by the Ethics Committee of Zibo Women & Children Hospital. The experimental procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Zibo Women & Children Hospital.
Not applicable.
The authors declare that they have no competing interests.
Overexpression of miR-26a-5p alleviates LPS-induced acute lung injury in mice. Mice in each group underwent intraperitoneal injection of miR-26a-5p mimics or NC mimics (20 mg/kg) 24 h before treatment with 5 mg/kg LPS. Mice were sacrificed after LPS administration for 24 h and then lung tissues were collected for analysis (except the survival experiment). (A) The expression of miR-26a-5p in lung tissues was detected by reverse transcription-quantitative PCR (n=5/group). (B) Pathological changes in the lung tissues observed by H&E staining (×100, magnification) (n=5/group). (C) The lung W/D weight ratio was assessed among the experimental groups (n=6/group). The activity of (D) MPO and the content of (E) MDA in lung tissues were detected using the corresponding test kits (n=6/group). (F) The survival rates were observed during 84 h following LPS treatment (n=10/group). Data were presented as the mean ± SD. **P<0.01 vs. Sham group; ##P<0.01 vs. LPS + mimic NC group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide; W/D, weight/dry; MDA, malondialdehyde; MPO, myeloperoxidase.
Overexpression of miR-26a-5p alleviates lung tissue inflammation in LPS-induced acute lung injury mice. Mice in each group underwent intraperitoneal injection of miR-26a-5p mimics or NC mimics (20 mg/kg) 24 h before treatment with 5 mg/kg LPS. And mice were sacrificed after LPS administration for 24 h and then BALF were collected for analysis. (A) The total protein concentration in BALF was measured by BCA method (n=6/group). The number of (B) neutrophils and (C) lymphocytes in BALF was determined using a hemocytometer (n=6/group). The expression level of (D) IL-1β, (E) IL-6 and (F) TNF-α in BALF was detected by ELISA (n=6/group). Data are presented as the mean ± SD. **P<0.01 vs. Sham group; ##P<0.01, vs. LPS + mimic NC group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide; BALF, bronchoalveolar lavage fluid.
Overexpression of miR-26a-5p decreases lung tissue cell apoptosis in LPS-induced acute lung injury mice. Mice in each group were intraperitoneal injection of miR-26a-5p mimics or NC mimics (20 mg/kg) 24 h before treatment with 5 mg/kg LPS. And mice were sacrificed after LPS administration for 24 h and then lung tissues were collected for analysis. (A) Apoptotic cells in the lung tissues were detected by TUNEL staining (magnification, ×400) (n=5/group). The mRNA expression of (B) Bcl-2, (C) Bax and (D) cleaved caspase-3 in the lung tissues was detected by reverse transcription-quantitative PCR (n=5/group). (E) Representative western blot bands of Bcl-2, Bax and cleaved caspase-3. (F) Semi-quantitative data for the level of (F) Bcl-2, (G) Bax and (H) cleaved caspase-3 expression (n=5/group). Data were presented as the mean ± SD. **P<0.01 vs. Sham group; ##P<0.01 vs. LPS + mimic NC group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide; TUNEL, Terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling.
Overexpression of miR-26a-5p decreases apoptosis and inflammatory responses in LPS-induced A549 cells. A549 cells were transfected with miR-26a-5p mimic or mimic NC (10 nM), and then treated with LPS for 24 h, followed by the assessment of cell apoptosis and inflammatory response. (A) The expression of miR-26a-5p was detected by reverse transcription-quantitative PCR in A549 cells treated with various concentrations (0, 5, 10 and 20 µg/ml) of LPS. (B) The expression of miR-26a-5p was detected in transfected A549 cells. (C) The cell viability was measured by MTT assay in transfected A549 cells. (D) Apoptotic cells were detected by flow cytometry in transfected A549. The expression levels of (E) IL-1β, (F) IL-6 and (G) TNF-α were detected by ELISA in transfected A549 cells. Data were presented as the mean ± SD. (A) *P<0.05, **P<0.01 vs. LPS (0 µg/ml) group. (B-G) **P<0.01 vs. Control group; ##P<0.01 vs. LPS + mimic NC group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide.
CTGF is the target gene of miR-26a-5p. (A) The binding target of miR-26a-5p and CTGF was predicted by TargetScan software 7.2. (B) Luciferase activity of CTGF-WT and CTGF-MT following the co-transfection of miR-26a-5p mimic or mimic NC in A549 cells detected by dual-luciferase reporter gene assay. (C) The mRNA expression of CTGF was detected by reverse transcription-quantitative PCR in A549 cells transfected with mimic NC or various concentrations (5, 10 and 20 nM) of miR-26a-5p. (D) The protein expression of CTGF was detected by western blotting in A549 cells transfected with mimic NC or various concentrations (5, 10 and 20 nM) of miR-26a-5p. Data were presented as the mean ± SD. (B) **P<0.01 vs. mimic NC group. (C and D) **P<0.01 vs. Control group; #P<0.05, ##P<0.01 vs. LPS + mimic NC group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide; CTGF, connective tissue growth factor; UTR, untranslated region; WT, wild-type; MT, mutant.
CTGF overexpression reverses the effect of miR-26a-5p on the apoptosis and inflammatory responses in LPS-induced A549 cells. A549 cells were co-transfected with miR-26a-5p mimic (10 nM), oe-CTGF (10 nM), mimic NC (10 nM) and oe-CTGF NC (10 nM), and then treated with LPS for 24 h, followed by the assessment of cell apoptosis and inflammatory response. (A) The mRNA expression of CTGF was detected by reverse transcription-quantitative PCR in transfected A549 cells. (B) The protein expression of CTGF was measured by western blotting in transfected A549 cells. (C) Cell viability was detected by MTT assay in transfected A549 cells. (D) The cell apoptosis was detected by flow cytometry in transfected A549 cells. The expression levels of (E) IL-1β, (F) IL-6 and (G) TNF-α were detected by ELISA in transfected A549 cells. Data were presented as the mean ± SD. (A and B) **P<0.01 vs. LPS + oe-CTGF NC group; ##P<0.01 vs. LPS + mimic NC + oe-CTGF NC group; &&P<0.01 vs. LPS + miR-26a-5p mimic + oe-CTGF NC or LPS + mimic NC + oe-CTGF group. (C-G) **P<0.01 vs. LPS + mimic NC + oe-CTGF NC group; ##P<0.01 vs. LPS + miR-26a-5p mimic + oe-CTGF NC or LPS + mimic NC + oe-CTGF group. miR, microRNA; NC, negative control; LPS, lipopolysaccharide; CTGF, connective tissue growth factor; oe, overexpression plasmid.