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. 2018;17(16):2001-2018.
doi: 10.1080/15384101.2018.1509635. Epub 2018 Sep 19.

MicroRNA-27a Alleviates LPS-induced Acute Lung Injury in Mice via Inhibiting Inflammation and Apoptosis Through Modulating TLR4/MyD88/NF-κB Pathway

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MicroRNA-27a Alleviates LPS-induced Acute Lung Injury in Mice via Inhibiting Inflammation and Apoptosis Through Modulating TLR4/MyD88/NF-κB Pathway

MinJie Ju et al. Cell Cycle. .
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Abstract

Acute lung injury (ALI) is a critical clinical condition with a high mortality rate, characterized with excessive uncontrolled inflammation and apoptosis. Recently, microRNAs (miRNAs) have been found to play crucial roles in the amelioration of various inflammation-induced diseases, including ALI. However, it remains unknown the biological function and regulatory mechanisms of miRNAs in the regulation of inflammation and apoptosis in ALI. The aim of this study is to identify and evaluate the potential role of miRNAs in ALI and reveal the underlying molecular mechanisms of their effects. Here, we analyzed microRNA expression profiles in lung tissues from LPS-challenged mice using miRNA microarray. Because microRNA-27a (miR-27a) was one of the miRNAs being most significantly downregulated, which has an important role in regulation of inflammation, we investigated its function. Overexpression of miR-27a by agomir-27a improved lung injury, as evidenced by the reduced histopathological changes, lung wet/dry (W/D) ratio, lung microvascular permeability and apoptosis in the lung tissues, as well as ameliorative survival of ALI mice. This was accompanied by the alleviating of inflammation, such as the reduced total BALF cell and neutrophil counts, decreased levels of tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-6) interleukin-1β (IL-1β) and myeloperoxidase (MPO) activity in BAL fluid. Toll-like receptor 4 (TLR4), an important regulator of the nuclear factor kappa-B (NF-κB) signaling pathway, was identified as a novel target of miR-27a in RAW264.7 cells. Furthermore, our results showed that LPS stimulation increased the expression of MyD88 and NF-κB p65 (p-p65), but inhibited the expression of inhibitor of nuclear factor-κB-α (IκB-α), suggesting the activation of NF-κB signaling pathway. Further investigations revealed that agomir-miR-27a reversed the promoting effect of LPS on NF-κB signaling pathway. The results here suggested that miR-27a alleviates LPS-induced ALI in mice via reducing inflammation and apoptosis through blocking TLR4/MyD88/NF-κB activation.

Keywords: Acute lung injury; TLR4/MyD88/NF-κB pathway; inflammation and apoptosis; microRNA-27a.

Figures

Figure 1.
Figure 1.
miR-27a was downregulated in LPS-induced acute lung injury in mice. Groups of mice (n = 10/group) were challenged with 1 mg/kg LPS or PBS for 24 h. Lung tissue samples, splenocytes and the BALF were collected and analyzed. (a) Lung tissues from each experimental group were processed for histological evaluation. Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control group. (b) Heat map of miRNA profiles represented the significantly regulated miRNAs (n = 3/group). (C-E) MiR-27a expression was validated by qRT-PCR in lung tissues, BALF and splenocytes of mice challenged with LPS (n = 3/group). Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control group.
Figure 2.
Figure 2.
Increased miR-27a ameliorated LPS induced ALI in mice. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h and then lung tissues were collected for analysis. (a) The miR-27a level in lung tissue samples were measured by qRT-PCR (n = 3/group). (b) Lung tissues from each experimental group were processed for histological evaluation (n = 3/group). (c, d) The Evans blue content and lung wet/dry assay (n = 3/group). (e) Oxygenation index (PaO2/FIO2) were determined (n = 3/group). Data represent the mean ± SD of three independent experiments. *p <  0.05, **p < 0.01 vs. control group. ##p < 0.01 vs. LPS alone group. (f) The survival rates were observed during 96 h after exposure to LPS (n = 10/group). **p < 0.01 vs. control group. ##p < 0.01 vs. LPS alone group.
Figure 3.
Figure 3.
Overexpression of miR-27a attenuated LPS-induced inflammatory response. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h, and then the BALF were collected for analysis. (a,b) The total counts of cells and neutrophils from the BALF were counted using a hemocytometer (n = 3/group). (c) MPO activity in lung tissues was measured at 24 h after LPS challenge (n = 3/group). (d-f) IL-6, IL-1β and TNF-α levels in BALF were measured at 24 h after LPS challenge (n = 3/group). Data represent the mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs. control group. ##p < 0.01 vs. LPS alone group.
Figure 4.
Figure 4.
Overexpression of miR-27a inhibited LPS-induced apoptosis. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h, and then the BALF were collected for analysis. (a) Apoptotic cells in the tissues were observed and the number of TUNEL positive cells was counted in 5 to 10 fields for each slide (n = 3/group). (b) The protein expression levels of Bcl-2, Bax and cleaved-caspase 9 were detected by Western Blot (n = 3/group). Data represent the mean ± SD of three independent experiments. *p <  0.05, **p < 0.01 vs. control group. ##p < 0.01 vs. LPS alone group. (C) The bands were semi-quantitatively analyzed by using Image J software, normalized to β-actin density. Data represent the mean ± SD of three independent experiments. *p  <  0.05, **p < 0.01 vs. control group. ##p < 0.01 vs. LPS + agomir-NC group.
Figure 5.
Figure 5.
TLR4 was a direct target of miR-27a. (a, b) The putative binding site of miR-27a and TLR4 is shown. (c) Luciferase assay of HEK293 cells co-transfected with firefly luciferase constructs containing the TLR4 wild-type or mutated 3´-UTRs and miR-27a mimics, mimics NC, miR-27a inhibitor or inhibitor NC, as indicated (n = 3). Data represent the mean ± SD of three independent experiments. **p < 0.01 vs mimics NC, ## p < 0.01 vs inhibitor NC. (d) The expression of TLR4 protein after transfection with miR-27a mimic or miR-27a inhibitor was measured by Western Blot. Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. inhibitor NC. ##p < 0.01 vs. mimics NC. (e) Expression of TLR4 was measured using Western Blot in lung tissues from ALI mice injection with agomir-27a or agomir NC (n = 3/group). Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control group. ##p < 0.01 vs. LPS + agomir-NC group. (f) Expression of TLR4 was measured using IHC in lung tissues from ALI mice injection with agomir-27a or agomir NC (n = 3).
Figure 6.
Figure 6.
miR-27a regulates the inflammatory response and apoptosis in LPS treated RAW 264.7 cells through targeting TLR4. RAW 264.7 cells were co-transfected with pcDNA-TLR4 and miR-27a mimics or treated with si-TLR4 alone, and then treated with LPS for 6  h, followed by the assessment of cell apoptosis and inflammatory response. (a-c, e-g) IL-6, IL-1β and TNF-α levels were measured using ELISA assay (n = 3). (d, h) Cell apoptosis was determined by flow cytometry. Data represent the mean ± SD of three independent experiments. *p < 0.05, **p 0.  0.01 vs. control group. ##p  *  0.01 vs. LPS alone group.
Figure 7.
Figure 7.
Overexpression of miR-27a blocked TLR4/NF-κB pathway in LPS-induced ALI mice. Groups of mice were given agomir-27a or agomir NC (2 mg/kg) by tail intravenous injection 24 h prior to 1 mg/kg LPS treatment. The mice were sacrificed after LPS administration for 24 h, and then the BALF were collected for analysis. (a) The levels of MyD88, nuclear p-p65 and p-IκB-α were measured by Western Blot (n = 3). (b) The bands were semi-quantitatively analyzed by using Image J software, normalized to β-actin density. Data represent the mean ± SD of three independent experiments. **p < 0.01 vs. control group. ##p < 0.01 vs. LPS + agomir-NC group. (c) Expression of nuclear p-p65 was measured using IHC in lung tissues from ALI mice injection with agomir-27a or agomir NC.
Figure 8.
Figure 8.
Scheme summarizing the protective effects of miR-27a on LPS-induced acute lung injury via the inhibiting TLR4/MyD88/NF-κB activation. LPS can induce NF-κB activation via TLR4-MyD88 signaling, IκBα acts as an inhibitor of NF-κB, Once the pathway is activated and IκBα is degraded, the NF-κB subunit p65 translocates from the cytoplasm to nucleus, which triggers the transcription of target genes, including TNF-α, IL-1β, and IL-6, and thus regulates inflammatory responses. However, miR-27a attenuates the release of pro-inflammatory cytokines by inhibiting TLR4/MyD88/NF-κB activation.

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