Male BALB/c mice (age, 6 weeks; weight, 18-22 g) were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. All mice were housed under standard conditions (12-h light-dark cycle at 25-27°C and ~40% humidity) with free access to food and water.

The mice were randomly divided into four groups (10 mice per group) as follows: i) The control group: 1.5 mg/kg normal saline (NS) was administered; ii) lipopolysaccharide (LPS) group: 0.2 ml of LPS (10 mg/kg, Escherichia coli 055:B5; Sigma-Aldrich; Merck KGaA) was intravenously administered via the tail vein; iii) LPS + agomir-129 group; and iv) LPS + agomir negative control (NC) group. In the mice in the agomir-129/agomir-NC group, in addition to LPS, 0.2 ml agomir-129/agomir-NC (8 mg/kg) was intravenously administered via the tail vein (20). Agomir-129 or agomir-NC were injected intravenously via the tail vein every third day until ALI was induced. Pentobarbital sodium (50 mg/kg, intraperitoneal injection) was used for anesthesia before each operation, and all efforts were made to minimize animal suffering. No mice were found dead during the anesthesia process. If an animal reached the defined humane endpoints [a >15% body weight loss in 1-2 days or an overall >20% loss in body weight or displayed obvious signs of suffering (lethargy, squinted eyes, dehydration or hunched back), it was humanely euthanized. Sacrifice was performed by an intraperitoneal injection of pentobarbital sodium (50 mg/kg) followed by cervical dislocation, and the death of the mice was confirmed by the cessation of respiration and heartbeat (21). In the survival experiment, the mice were observed for 7 days after the LPS injection, while other mice were observed for 3 days after the LPS injection. All mice were humanely sacrificed when they reached the humane endpoint or the set experimental endpoint. The lungs were then excised, intact. Subsequently, the trachea was lavaged with 1 ml of sterile ice-cold PBS and bronchoalveolar lavage fluid (BALF) was collected. BALF was pooled and centrifuged at 200 × g for 10 min at 4°C, and the harvested supernatant was stored at −80°C until mediator analysis.

In addition, the survival experiments were performed in 4 groups of mice (n=10/group) (LPS, Control, LPS + agomir-129, LPS + agomir-NC). The survival rate was defined from 0 to 7 days following treatment and analyzed with the Kaplan-Meier survival analysis and the log rank test. All animal experiments were approved by the Animal Care and Use Committee of Henan Provincial People's Hospital (approval no. 2019-00112).

miRNA data (accession no. GSE133733) were downloaded from open microarray datasets, deposited in NCBI's GEO database (http://www.ncbi.nlm.nih.gov/geo/). The miRNA array expression data were analyzed with the Qlucore Omics Explorer (QOE 3.1) bioinformatics software (http://www.qlucore.com/).

RAW 264.7 cells (cat. no. ATCC^® TIB-71) were obtained from ATCC and cultured in DMEM/F12 supplemented with 10% FBS (Abcam) at 37°C and in a 5% CO₂ atmosphere. RAW264.7 cells (2×10⁵ cells) were seeded in a 6-well plate and transfected with 100 nM of miR-129 mimics/inhibitor at 37°C for 48 h using Lipofectamine^® 2000 (Invitrogen; Thermo Fisher Scientific, Inc.). miR-129 mimic/inhibitor and their NC oligonucleotides (NC mimic or NC inhibitor) were obtained from Shanghai GenePharma Co., Ltd. The sequences were as follows: miR-129 mimic, 3′-CUUUUUGCGGUCUGGGCUUGC-5′; miR-129 inhibitor, 3′-GCAAGCCCAGACCGCAAAAAG-5′; mimic NC, 5′-UUUGUACUACACAAAAGUACUG-3′; inhibitor NC, 5′-UUCUCCGAACGUGUCACGUTT-3′. The cells were harvested at 48 h following transfection for testing.

A series of ALI cell models have been established to simulate the status of ALI caused by diverse pathogeneses. The human type II lung epithelial A549 cell line is usually used to establish cell apoptosis models in LPS-induced ALI (22,23). Among these, RAW 264.7 macrophages are usually used to establish cell inflammation responses models in LPS-induced ALI (24,25). Therefore, RAW cells were selected for use in ex vivo experiments as the focus of the present study was on the inflammatory responses in ALI. For this purpose, RAW 264.7 cells were co-transfected with miR-129 mimics (100 nmol/l), pcDNA-TAK1 (2 μg), or si-TAK1 (100 nmol/l) for 48 h using Lipofectamine 3000^® (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in 5% CO₂, followed by LPS stimulation (100 ng/ml) for a further 6 h, as previously described (26). Subsequently, they were cultured in complete medium for 48 h, in order to perform the subsequent experiments. A TAK1 expression vector was constructed through the insertion of the full sequence of TAK1 into the pcDNA 3.1 vector (Invitrogen; Thermo Fisher Scientific, Inc.). Specific small interfering RNAs (siRNAs) for TAK1 (5′-CATCCAGTGCCAAGCAGCTCATATT-3′) and si-Scramble (5′-CATGAGTCCAACGGATCACTCCATT-3′) were purchased from Shanghai GenePharma Co., Ltd. RAW 264.7 cells co-transfected with the pcDNA-vector and si-Scramble were used as a control group. Agomir-129 and agomir-NC were synthesized by GenePharma Co., Ltd. The agomiR-129 and agomiR-NC, whose sequence was the same as the miR-129 mimics and mimics NC, were modified with methylation, cholesterol addition and thiophosphorylation, in order to improve the stability and transfection efficiency and to simulate miRNA activity in the animal body (27).

Total RNA was isolated from the lung tissues using the RNeasy Mini kit (Qiagen GmbH). Reverse transcripts of miR-129 and TAK1 were synthesized using the MicroRNA Reverse Transcription kit (Thermo Fisher Scientific, Inc.) and the PrimeScript RT reagent kit (Takara Bio, Inc.), respectively. miR-129 and TAK1 expression was measured with SYBR-Green (Beijing Solarbio Science & Technology Co., Ltd.) on a Light Cycler instrument (Bio-Rad Laboratories, Inc.). The following primers were used for RT-qPCR analysis: miR-129 forward, 5′-GTTGGGGAGATTTAGTTTGTT-3′ and reverse, 5′-CCTACTCCAATTCCCCCTATAATAC-3′; U6 forward, 5′-CTCGCTTCGGCAGCACA-3′ and reverse, 5′-AACGCTTCACGAATTTGCGT-3′; TAK1 forward, 5′-GATATCCTGTCGACAGCCTCCGC-3′ and reverse, 5′-AACGTAACGGGCCCAGAGAA-3′; GAPDH forward, 5′-GTGGTGAAGACGCCAGTGGA-3′ and reverse, 5′-CGAGCCACATCGCTCAGACA-3′. The thermocycling conditions were as follows: 50°C for 2 min and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 10 min. Relative miRNA expression was analyzed using the 2^−ΔΔCq method (28).

Mouse lungs from all groups were fixed with 10% formalin and micro-sectioned to a thickness of 5 μm using a Leica SM2010R microtome (Leica Microsystems, Inc.). The tissues were embedded in paraffin and then subjected to hematoxylin and eosin (H&E) staining. Images were viewed and captured by a Nikon E-800M microscope (Nikon Corporation) at ×200 magnification. Inflammatory cell infiltration, bleeding and interstitial and alveolar edema were observed under a light microscope, according to the following lung injury score: 0, normal pulmonary appearance; 1, slight injury, mild interstitial congestion and neutrophil leukocyte infiltrations; 2, moderate injury: Moderate interstitial congestion and neutrophil leukocyte infiltrations; 3, severe injury, perivascular edema formation, partial leukocyte infiltration, moderate neutrophil leukocyte infiltration; 4, very severe histological injury: Severe destruction of the lung architecture and massive neutrophil leukocyte infiltration.

The Evans blue (EB) (Sigma-Aldrich; Merck KGaA) dye extravasation method was used to assess pulmonary permeability, as previously described (29). The dye concentration was reported as the absorbance relative to the weight of dry lung tissue (μg/100 mg dry tissue).

Following a 72-h LPS challenge, the mice were sacrificed. The lungs were excised, and the right lungs were weighed and dried in an incubator at 55°C. After 60 h, the lungs were weighed again to calculate the wet-to-dry (W/D) ratio.

Murine cytokine-specific Quantikine ELISA kits (R&D Systems Europe, Ltd.) were used to detect the IL-6, IL-1β, TNF-α, Cxcl2, JE (the murine homolog of human CCL2) and KC (the murine homolog of human IL-8) levels in BALF, as previously described (14).

The lung tissue sections were deparaffinized with xylene, rehydrated with ethanol at graded concentrations of 70-100% (v/v) and washed with water. The sections were then treated with 100 μl proteinase K (20 μg/ml, Roche Diagnostics) for 15 min, at room temperature. After the sections were washed three times with PBS, they were stained with prepared terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) solution, using the in situ Cell Death Detection kit (Roche Diagnostics). Cell quantification was performed under an inverted fluorescence microscope (DP73; Olympus Corporation) at ×200 magnification. TUNEL-positive cells were counted in three fields of view per section.

The miRNA target prediction tools PicTar version 2007 (https://pictar.mdc-berlin.de/) and TargetScan Release 7.0 (http://targetscan.org/) were used to search for putative targets of miR-153.

The 3′-UTR of TAK1 and its mutated sequence were inserted into the pGL3 control vector (Promega Corporation) to construct the wild-type (wt) TAK1-3′-UTR vector and mutant TAK1-3′-UTR vector, respectively. RAW 264.7 cells were seeded in 24-well plates (5.0×10⁵/well) and were transfected with either the wild-type or mutant reporter vector (2 μg), combined with the miR-129 mimics/inhibitor (100 nM), using Lipofectamine 2000^® (Invitrogen, Thermo Fisher Scientific Inc.). The Renilla luciferase expression vector pRL-TK (Promega Corporation) was employed as the reference. At 48 h post-transfection, luciferase activity was detected using the dual luciferase reporter kit (Beyotime Institute of Biotechnology).

The expression of cleaved caspase-3, TAK1 and p-p65 in the lung sections was evaluated by immunohistochemical staining, as previously described (30), with the following primary antibodies diluted to 1:200: Cleaved caspase-3 (cat no. 9664, Cell Signaling Technology, Inc.), TAK1 (cat no. 5206, Cell Signaling Technology, Inc.), and p-p65 (cat no. 3036, Cell Signaling Technology, Inc.). Samples were photographed under a Leica DMD 108 light microscope. Immunohistochemical staining was independently evaluated by two observers, and the immunohistochemistry images of lung tissue samples were scored as previously described (31).

Total protein was extracted using the RIPA buffer. The extraction and isolation of nuclear and cytoplasmic proteins were performed with the Cytoplasmic and Nuclear Protein Extraction kit (cat no. 78833, Thermo Fisher Scientific, Inc.), according to the manufacturer's instructions. The protein concentration was determined using the bicinchoninic acid assay (Pierce, Thermo Fisher Scientific Inc.), following centrifugation of the protein fraction at 10,000 × g for at 4°C for 15 min. Proteins (20-50 μg) were separated by 12% SDS-PAGE (w/v) and transferred onto PVDF membranes (EMD Millipore). The membranes were then blocked with 1% BSA for 2 h at room temperature, and incubated with the following primary antibodies overnight at 4°C: TAK1 (cat no. 5206, 1:1,000), Bax (cat no. 5023, 1:1,000), Bcl-2 (cat no. 3498, 1:1,000), nuclear p-p65 (cat no. 3036, 1:1,000), p-IκB-α (cat no. 2859, 1:1,000), IκB-α (cat no. 4814, 1:1,000), histone H3 (cat no. 9728, 1:1,000) and β-actin (cat no. 4970, 1:1,000). Subsequently, the membranes were incubated with HRP-linked anti-rabbit IgG antibody (cat no. 7074, 1:2,000) and incubated with ECL reagent (GE Healthcare) for the detection of protein expression. All antibodies were obtained from Cell Signaling Technology, Inc. The gray value of the analyzed proteins was determined using the ImageJ software (version 1.46; Rawak Software Inc.).

GraphPad Prism 5.0 (GraphPad Software, Inc.) was used to perform the statistical analyses. Statistically significant differences were analyzed using an unpaired Student's t-test or one-way analysis of variance followed by Tukey's post hoc test. The correlation between TAK1 and miR-129 expression was analyzed using Pearson's correlation analysis. The differences in overall survival were assessed by Kaplan-Meier survival analysis and the log-rank test. All data are presented as the mean ± SD, and P<0.05 was considered to indicate a statistically significant difference.

The analysis of the expression of miRNAs included in the miRNA array expression profile dataset (GSE133733) from GEO revealed that there was a significant difference between the control group and the sepsis group (Fig. 1A). Out of the 38 miRNAs with a notable difference in relative expression levels, 21 were downregulated and 17 were upregulated in the sepsis group, compared with the control group: The expression of miR-129-5p (32), miR-27a (33), and miR-150 (34) was decreased, while the expression of miR-34b-5p (35), miR-155-3p (36) and miR-146-3p (37) was increased. This finding is consistent with previous reports and indicates the reliability of the microarray results in the present study. However, miR-129 was selected for further investigation as it exhibited the most statistically significant difference between the two groups and as previous studies have discussed the role of miR-129 in regulating injury and inflammatory response in different organ injury models (19,38,39).

To evaluate the therapeutic effects of miR-129, a model of LPS-induced lung injury was used, as previously described (40), as LPS administration is one of the most widely used murine experimental models for lung injury (41,42). As depicted in Fig. 1B, the administration of LPS alone resulted in the destruction of the lung architecture and in inflammation. The lung injury scores based on histopathological changes were significantly higher in the LPS group than in the control group, indicating that the ALI model was successfully established. At 72 h after the LPS challenge, lung tissue and BALF were harvested, and RT-qPCR was performed in order to measure the miR-129 expression levels. As was expected, miR-129 expression was notably decreased in response to the LPS challenge in BALF and lung tissues (Fig. 1C and D). These findings indicate that miR-129 may be involved in the development of ALI.

Gain-function experiments were performed through the administration of agomiR-129, in order to investigate the potential protective effects of miR-129 against LPS-induced ALI. The expression levels of miR-129 in lung tissues was significantly elevated following the agomiR-129 injection in comparison with the control group expression levels (Fig. 2A), suggesting miR-129 overexpression has been successfully induced in mice, following agomiR-129 administration. Moreover, the miR-129 expression levels were significantly decreased in the LPS group as compared with the control group, whereas in the LPS + agomiR-129 group, the level of miR-129 was significantly increased compared with the LPS + agomiR-NC group (Fig. 2A). Histopathological analysis revealed that agomiR-129 administration attenuated the severity of lung injury caused by LPS, as indicated by the decrease in lung injury scores (Fig. 2B). The lung W/D ratio was calculated as an indicator of lung edema, which is an important feature of ALI (43). These results demonstrated that the lung W/D ratio was markedly increased by the LPS challenge, whereas it was significantly decreased following agomiR-129 administration in mice with LPS-induced ALI (Fig. 2C). In addition, the evaluation of lung microvascular permeability with the EB extravasation method revealed that the LPS challenge led to a significant increase in EB extravasation, which is considered as an indicator of lung vascular permeability, as compared with the control group EB extravasation (Fig. 2D). As was expected, agomiR-129 administration effectively reduced the LPS-induced increase in EB extravasation. Furthermore, agomiR-129 administration resulted in a significant increase in the survival rate (P<0.01; Fig. 2E). Taken together, these findings suggest that miR-129 may alleviate the pathological effects of ALI.

The effects of miR-129 on the inflammatory response in ALI were then examined. The measurement of the levels of pro-inflammatory cytokines and chemokines in BALF from mice with ALI revealed that the concentrations of the inflammatory cytokines, IL-1β, IL-6 and TNF-α, and the chemokines, JE, Cxcl2 and KC, were markedly higher in the LPS group than in the control group. However, agomiR-129 administration inhibited the LPS-induced inflammatory cytokine and chemokine production (Fig. 3). These results indicate that miR-129 upregulation alleviates LPS-induced inflammatory response in ALI.

Since alveolar epithelial cells apoptosis is another important feature of ALI, lung tissue apoptosis was evaluated by TUNEL assay. The number of TUNEL-positive cells was markedly increased in the mouse lungs following LPS administration; however, the number of TUNEL-positive cells was markedly decreased in the lungs of the mice that were administered agomir-129 after the LPS administration (Fig. 4A). In addition, the relative levels of important proteins involved in apoptotic pathways were measured. Immunohistochemistry assay demonstrated that the expression levels of cleaved caspase-3 were markedly higher in the LPS group than in the control group, whereas agomiR-129 administration markedly inhibited the LPS-induced increase in caspase-3 expression levels (Fig. 4B). In addition, western blot analysis revealed that in comparison with the control group, the protein levels of the pro-apoptotic proteins, cleaved caspase-3 and Bax, were significantly increased, while the protein levels of the anti-apoptotic protein, Bcl-2, were decreased in the LPS group. AgomiR-129 administration markedly attenuated the effects of LPS on the apoptosis-related protein levels (Fig. 4C and D). Collectively, these results suggest that miR-129 upregulation inhibits LPS-induced lung cell apoptosis in mice.

In order to elucidate the molecular mechanisms instigating the miR-129 protective effects against LPS-induced apoptosis and the inflammatory response in mice with ALI, the potential target genes of miR-129 were analyzed using PicTar version 2007 (https://pictar.mdc-berlin.de/) and TargetScan Release 7.0 (http://targetscan.org/). Bioinformatics analysis indicated that TAK1 was a miR-129 potential target (Fig. 5A). To verify this association, a luciferase reporter assay was performed. To this end, the transfection efficiency of miR-129 mimics/inhibitor was first evaluated by RT-qPCR. The results revealed that miR-129 relative expression was significantly upregulated/downregulated as compared with the mir-NC group (Fig. 5B). Subsequently, it was found that the luciferase activity of wt-TAK1 3′UTR was significantly decreased after miR-129 mimics transfection and increased after the miR-129 inhibitor transfection. However, this effect was not observed, when mut-TAK1 3′UTR was used (Fig. 5C). It was also observed that miR-129 overexpression resulted in a decrease in TAK1 expression in RAW 264.7 cells, while miR-129 knockdown resulted in a significant increase in TAK1 protein expression levels (Fig. 5D).

In order to determine whether miR-129 also regulates TAK1 expression in vivo, TAK1 expression was detected in the lungs of mice with ALI. The results revealed that TAK1 expression was significantly increased following the LPS challenge (Fig. 5E). Furthermore, miR-129 expression was found to inversely correlate with TAK1 protein expression levels in lung tissues (Fig. 5F). As also observed in vitro, the increase in TAK1 protein expression due to LPS administration was markedly attenuated by the agomiR-129 administration (Fig. 5G). On the whole, these results indicate that TAK1 is a functional target of miR-129 in ALI.

Considering the important role of TAK1 in the inflammatory response following ALI, the present study attempted to determine whether TAK1 mediated the protective effects of miR-129 against ALI-associated inflammation (44). A TAK1 expression vector, pcDNA-TAK1, or si-TAK1, together with miR-129 mimics, was transfected into RAW264.7 cells 6 h prior to LPS stimulation. The results demonstrated that pcDNA-TAK1 caused a significant increase in TAK1 protein expression levels, while si-TAK1 transfection resulted in a marked decrease in TAK1 protein expression levels (Fig. 6A). The production of the inflammatory cytokines, IL-1β, IL-6 and TNF-α was evaluated using ELISA in LPS-stimulated RAW264.7 cells co-transfected with pcDNA-TAK1 and miR-129 mimics or si-TAK1 and miR-129 mimics. As depicted in Fig. 6B-D, LPS stimulation significantly increased the secretion of IL-1β, IL-6 and TNF-α, which was suppressed by si-TAK1, whereas it was enhanced by TAK1 overexpression. In LPS-stimulated RAW264.7 cells, transfection with si-scramble or pcDNA-vector alone had no significant effect on the inflammatory cytokine levels when compared with the LPS group. Notably, the inhibitory effects of miR-129 mimics on the production of the inflammatory cytokines were reversed by TAK1 overexpression in LPS-stimulated RAW264.7 cells Thus, these findings imply that miR-129 protects RAW264.7 cells from LPS-induced inflammatory response by targeting TAK1.

TAK1 has been shown to activate the NF-κB pathway (45), and the inhibition of the TAK1/NF-κB signaling pathway has been found to reduce the production of the pro-inflammatory cytokines, IL-1β, IL-6 and TNF-α (46). In the present study, in order to investigate the effects of miR-129 on TAK1/NF-κB pathway activation, the p-IKKβ, IKKβ, p-IκB-α, and IκB-α expression levels of were examined by western blot analysis. It was found that the TAK1, p-IKKβ and p-IκB-α protein expression levels were significantly increased after the LPS challenge, and that these effects were attenuated by agomiR-129 administration (Fig. 7). To confirm that miR-129 blocks the NF-κB pathway, the expression of nuclear p-p65 was examined by western blot analysis and immunohistochemistry. As was expected, the increased expression of nuclear p-p65 induced by LPS was reduced after agomiR-129 administration (Fig. 8). All these data indicate that miR-129 upregulation may block TAK1-mediated activation of the NF-κB pathway in mice with ALI.