NLRP3 Inflammasome Activation by MicroRNA-495 Promoter Methylation May Contribute to the Progression of Acute Lung Injury

Abstract

Acute lung injury (ALI) is a pulmonary disorder that causes acute respiratory failure, thus leading to relative high mortality worldwide. However, the molecular mechanisms of ALI remain largely unknown. MicroRNA (miRNA)-dependent control of gene expression at a post-transcriptional level has been recently reported. Herein, we identify a candidate miRNA, miR-495, that affects the progression of ALI. Alveolar macrophages (NR8383) were treated with 1 μg/mL lipopolysaccharide (LPS) to establish a cell-injury model. Combined with the data from western blot, methylation-specific PCR, methylated DNA immunoprecipitation, and chromatin immunoprecipitation assays, NLRP3 inflammasome activation and methylation-dependent repression of miR-495 were found in LPS-exposed NR8383 cells. Dual-luciferase reporter gene assay and miR-495 gain-of-function experiments confirmed that NLRP3 was a target of miR-495. Next, the expression of miR-495 and NLRP3 was overexpressed or silenced to assess their effects on NLRP3 inflammasome activation, alveolar macrophage inflammation, and pyroptosis in vitro. As demonstrated, overexpressed miR-495 alleviated alveolar macrophage inflammation and pyroptosis and inhibited NLRP3 inflammasome activation by negatively regulating the NLRP3 gene. Consistently, elevated miR-495 alleviated lung injury and reduced the neutrophil infiltration and inflammation in rat models of LPS-induced ALI. Taken together, the data in our study demonstrated that methylation of the miR-495 promoter could downregulate miR-495, whose elevation could attenuate the activation of the NLRP3 inflammasome to protect against ALI, which provides novel therapeutic targets for ALI treatment.

Keywords: NOD-like receptor family pyrin domain containing 3; acute lung injury; inflammasome; methylation; microRNA-495.

Figure 1

LPS-Induced ALI Exhibits Activated NLRP3 Inflammasome and Inflammation of Alveolar Macrophages in LPS-Induced ALI Is Inhibited with the Silencing of NLRP3 (A) Protein expression levels of NLRP3, caspase-1, ASC, IL-1β, IL-18, and Cle-GSDMD in NR8383 cells treated with F-12K culture medium or LPS as detected by western blot analysis. (B) Expression levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) and anti-inflammatory factor IL-10 in NR8383 cells treated with F-12K culture medium or LPS as detected by ELISA. (C) Protein expression levels of NLRP3, caspase-1, ASC, IL-1β, IL-18, and Cle-GSDMD in LPS-exposed NR8383 cells following transfection with sh-NLRP3 or sh-NC as measured by western blot analysis. (D) Expression levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) and anti-inflammatory factor IL-10 in LPS-exposed NR8383 cells following transfection with sh-NLRP3 or sh-NC as measured by ELISA. (E) Pyroptosis of LPS-exposed NR8383 cells following transfection with sh-NLRP3 or sh-NC, as determined by PI/Hoechst 33342 double staining. *p < 0.05 versus NR8383 cells treated with F-12K culture medium; ^#p < 0.05 versus LPS-exposed NR8383 cells transfected with sh-NC. An unpaired t test was used to analyze data expressed as mean ± SD between two groups if the data conformed to normal distribution and homogeneity of variance. Data among multiple groups were compared using one-way ANOVA followed by Tukey’s post hoc test. The experiment was repeated three times.

Figure 2

miR-495 Directly Targets NLRP3 (A) Binding site between miR-495 and NLRP3 3′ UTR predicted using the bioinformatic website microRNA.org. (B) Luciferase activity of NLRP3-WT and NLRP3-Mut following the co-transfection of miR-495 mimic or mimic NC detected by dual-luciferase reporter gene assay. (C) Expression of miR-495 in response to transfection with miR-495 mimic or mimic NC as measured by qRT-PCR. (D) Expression of NLRP3 in response to transfection with miR-495 mimic or mimic NC as measured by western blot analysis. (E) Protein expression of NLRP3 in response to transfection with hsa-miR-495 mimic or mimic NC as measured by western blot analysis. (F) Expression of NLRP3 in response to transfection with hsa-miR-495 mimic or mimic NC determined by qRT-PCR. *p < 0.05 versus NR8383 cells treated with mimic NC. Measurement data were expressed as mean ± SD. An unpaired t test was used to analyze data expressed as mean ± SD between two groups if the data conformed to normal distribution and homogeneity of variance. The experiment was repeated three times to obtain the mean value.

Figure 3

Expression of miR-495 Is Reduced in LPS-Exposed NR8383 Cells due to the Methylation of miR-495 Promoter (A) CpG island in the miR-495 gene promoter region predicted using the MethPrimer website. (B) Methylation of the miR-495 promoter in NR8383 cells treated with F-12K culture medium, LPS, combined LPS and DMSO, or combined LPS and 5-Aza as measured by MSP assay. (C) Enrichment of DNMT1 and DNMT3a/b in the miR-495 promoter region in NR8383 cells treated with F-12K culture medium, LPS, combined LPS and DMSO, or combined LPS and 5-Aza as measured by ChIP assay. (D) Expression of miR-495 in NR8383 cells treated with F-12K culture medium, LPS, combined LPS and DMSO, or combined LPS and 5-Aza as determined by qRT-PCR. (E) Methylation of miR-495 promoter in NR8383 cells treated with F-12K culture medium, LPS, combined LPS and DMSO, or combined LPS and 5-Aza detected by MeDIP assay. *p < 0.05 versus NR8383 cells treated with F-12K culture medium; ^#p < 0.05 versus NR8383 cells treated with LPS and DMSO. Measurement data were expressed as mean ± SD, and data among multiple groups were compared using one-way ANOVA and subjected to Tukey’s post hoc test. The experiment was repeated three times to obtain the mean value.

Figure 4

Activation of NLRP3 Inflammasome Triggered LPS Is Attenuated by Elevation of miR-495 (A) Expression of miR-495 in NR8383 cells treated with LPS, F-12K culture medium, combined LPS and mimic NC, or combined LPS and miR-495 mimic as detected by qRT-PCR. (B) Expression levels of NLRP3, ASC, and caspase-1 in NR8383 cells treated with LPS, F-12K culture medium, combined LPS and mimic NC, or combined LPS and miR-495 mimic as detected by western blot analysis. (C) Expression levels of IL-1β, IL-18, and Cle-GSDMD in NR8383 cells treated with LPS, F-12K culture medium, combined LPS and mimic NC, or combined LPS and miR-495 mimic as detected by western blot analysis. (D) Expression levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) and anti-inflammatory factor IL-10 in NR8383 cells treated with LPS, F-12K culture medium, combined LPS and mimic NC, or combined LPS and miR-495 mimic as detected by ELISA. *p < 0.05 versus NR8383 cells treated with F-12K culture medium; ^#p < 0.05 versus NR8383 cells treated with both LPS and mimic NC. Measurement data were expressed as mean ± SD, and data among multiple groups were compared using one-way ANOVA and subjected to Tukey’s post hoc test. The experiment was repeated three times to obtain the mean value.

Figure 5

Pyroptosis of Alveolar Macrophages Triggered by LPS Is Attenuated with the Elevation of miR-495 (A) Pyroptosis of LPS-exposed NR8383 cells determined by flow cytometry. (B) Pyroptosis of LPS-exposed NR8383 cells determined by PI/Hoechst 33342 double staining (200×). *p < 0.05, versus NR8383 cells treated with F-12K culture medium; ^#p < 0.05, versus LPS-exposed NR8383 cells transfected with mimic NC. Measurement data were expressed as mean ± SD, and data among multiple groups were compared using one-way ANOVA and subjected to Tukey’s post hoc test. The experiment was repeated three times to obtain the mean value.

Figure 6

Elevated Expression of miR-495 Depletes the Expression of NLRP3, Thus Hindering the LPS-Induced Inflammation In Vitro (A) Expression of miR-495 in LPS-exposed NR8383 cells in response to transfection with plasmids including mimic NC, miR-495 mimic, oe-NC, and oe-NLRP3, as determined by qRT-PCR. (B) Expression levels of NLRP3, ASC, and caspase-1 in NR8383 cells in response to transfection with plasmids including mimic NC, miR-495 mimic, oe-NC, and oe-NLRP3, as determined by western blot analysis. (C) Expression levels of L-1β and IL-18 in NR8383 cells in response to transfection with plasmids including mimic NC, miR-495 mimic, oe-NC, and oe-NLRP3, as determined by western blot analysis. (D) Expression levels of proinflammatory factors (TNF-α, IL-6, IL-1β, and IL-18) and anti-inflammatory factor IL-10 in NR8383 cells in response to transfection with plasmids including mimic NC, miR-495 mimic, oe-NC, and oe-NLRP3, as determined by ELISA. *p < 0.05 versus NR8383 cells treated with F-12K culture medium; ^#p < 0.05, versus LPS-exposed NR8383 cells transfected with mimic NC; ^&p < 0.05 versus LPS-exposed NR8383 cells co-transfected with miR-495 mimic and oe-NC. Measurement data were expressed as mean ± SD, and data among multiple groups were compared using one-way ANOVA and subjected to Tukey’s post hoc test. The experiment was repeated three times to obtain the mean value.

Figure 7

Elevated Expression of miR-495 Depletes the Expression of NLRP3, Thus Protecting against ALI In Vivo Rats were treated with normal saline or LPS, and LPS-treated rats were further introduced with NC agomir or miR-495 agomir. (A) Expression of miR-495 in lung tissues of rats as determined by qRT-PCR. (B) Expression levels of NLRP3, ASC, and caspase-1 in lung tissues of rats measured using western blot analysis. (C) Immunohistochemistry detection of NLRP3 contents in rat lung tissues (400×). (D) Pathological changes of lung tissues in rats observed using H&E staining (400×). (E) MPO activity in lung tissues of rats. (F) Detection of lung W/D ratio in rats. (G) Total protein content in BALF in rats. (H) Levels of proinflammatory factors (TNF-α, IL-6, and IL-1β) and anti-inflammatory factor IL-10 in the BALF of rats detected by ELISA. *p < 0.05 versus rats treated with normal saline; ^#p < 0.05 versus rats treated with LPS and NC agomir. Measurement data were expressed as mean ± SD, and data among multiple groups were compared using one-way ANOVA and subjected to Tukey’s post hoc test. The experiment was repeated three times to obtain the mean value.