circRNA_0001679/miR-338-3p/DUSP16 axis aggravates acute lung injury

Abstract

Acute lung injury (ALI) is a respiratory disorder characterized by acute respiratory failure. circRNA mus musculus (mmu)-circ_0001679 was reported overexpressed in septic mouse models of ALI. Here the function of circ_0001679 in sepsis-induced ALI was investigated. In vitro models and animal models with ALI were, respectively, established in mouse lung epithelial (MLE)-12 cells and C57BL/6 mice. Pulmonary specimens were harvested for examination of the pathological changes. The pulmonary permeability was examined by wet-dry weight (W/D) ratio and lung permeability index. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-1β in the bronchoalveolar lavage fluid (BALF), the lung tissues, and the supernatant of MLE-12 cells were measured by enzyme linked immunosorbent assay . Apoptosis was determined by flow cytometry. Bioinformatics analysis and luciferase reporter assay were used to assess the interactions between genes. We found that circ_0001679 was overexpressed in lipopolysaccharide (LPS)-stimulated MLE-12 cells. circ_0001679 knockdown suppressed apoptosis and proinflammatory cytokine production induced by LPS. Moreover, circ_0001679 bound to mmu-miR-338-3p and miR-338-3p targeted dual-specificity phosphatases 16 (DUSP16). DUSP16 overexpression reversed the effect of circ_0001679 knockdown in LPS-stimulated MLE-12 cells. Furthermore, circ_0001679 knockdown attenuated lung pathological changes, reduced pulmonary microvascular permeability, and suppressed inflammation in ALI mice. Overall, circ_0001679 knockdown inhibits sepsis-induced ALI progression through the miR-338-3p/DUSP16 axis.

Keywords: DUSP16; circ_0001679; miR-338-3p; sepsis.

Figure 1

Circ_0001679 knockdown inhibits cell apoptosis and inflammation. (a and b) RT-qPCR analysis of circ_0001679 expression in MLE-12 cells under the indicated treatment. (c–e) ELISA of the concentrations of IL-1β, IL-6, and TNF-α in MLE-12 cells under the indicated treatment. (f) Flow cytometry analysis of the apoptosis of MLE-12 cells under the indicated treatment. (g) Western blot analysis of the Bax and Bcl-2 protein levels in MLE-12 cells under the indicated treatment. Data are shown as the mean value ± SD. The experiments were repeated three times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 2

Circ_0001679 binds to miR-338-3p. (a) RT-qPCR analysis of the expression of candidate miRNAs in LPS-treated MLE-12 cells. (b) RT-qPCR analysis of the transfection efficiency of miR-338-3p mimics in MLE-12 cells. (c) The binding site of miR-338-3p in circ_0001679 (chr8:64811729-64811750[−]) predicted in starBase. (d) Luciferase reporter assay of the binding ability of miR-338-3p to circ_0001679. Data are shown as the mean value ± SD. The experiments were repeated three times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 3

DUSP16 is targeted by miR-338-3p. (a) RT-qPCR analysis of the expression of candidate targets in LPS-treated MLE-12 cells. (b) The binding site of miR-338-3p in DUSP16 3'-UTR predicted in TargetScan. (c) Luciferase reporter assay of the binding ability of miR-338-3p to DUSP16. (d) Western blot analysis of the DUSP16 protein level in MLE-12 cells under the indicated transfection. Data are shown as the mean value ± SD. The experiments were repeated three times. **p < 0.01 and ***p < 0.001.

Figure 4

Circ_0001679 promotes cell apoptosis and inflammatory response by upregulating DUSP16. (a) Western blot analysis of DUSP16 expression in MLE-12 cells after transfection with pcDNA3.1/DUSP16. (b–d) ELISA of the concentrations of IL-1β, IL-6, and TNF-α in MLE-12 cells under the indicated treatment. (e) Flow cytometry analysis of the apoptosis of MLE-12 cells under the indicated treatment. (f) Western blot analysis of the Bax and Bcl-2 protein levels in MLE-12 cells under the indicated treatment. Data are shown as the mean value ± SD. The experiments were repeated three times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Circ_0001679 knockdown alleviates lung injury and reduces inflammation in ALI mice. (a) The histological changes in lungs of ALI mice and sham mice were analyzed by hematoxylin and eosin staining. (b) Lung injury scores were analyzed and recorded. (c) Lung W/D ratio of mice and (d) lung permeability index in the lung tissues. (e–g) ELISA of the concentrations of IL-1β, IL-6, and TNF-α in the BALF and the lung tissues in each group. (h) Western blot analysis of the Bax and Bcl-2 protein levels in the lung tissues in each group. RT-qPCR analysis of (i) circ_0001679 expressions, (j) DUSP16 expression, and (k) miR-338-3p expression in the lung tissues in each group. (l–n) Pearson correlation analysis of the expression relationship between circ_0001679, DUSP16, and miR-338-3p in ALI mouse lung tissues. N = 12 per group. Data are shown as the mean value ± SD. The experiments were repeated three times. *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 5

Circ_0001679 knockdown alleviates lung injury and reduces inflammation in ALI mice. (a) The histological changes in lungs of ALI mice and sham mice were analyzed by hematoxylin and eosin staining. (b) Lung injury scores were analyzed and recorded. (c) Lung W/D ratio of mice and (d) lung permeability index in the lung tissues. (e–g) ELISA of the concentrations of IL-1β, IL-6, and TNF-α in the BALF and the lung tissues in each group. (h) Western blot analysis of the Bax and Bcl-2 protein levels in the lung tissues in each group. RT-qPCR analysis of (i) circ_0001679 expressions, (j) DUSP16 expression, and (k) miR-338-3p expression in the lung tissues in each group. (l–n) Pearson correlation analysis of the expression relationship between circ_0001679, DUSP16, and miR-338-3p in ALI mouse lung tissues. N = 12 per group. Data are shown as the mean value ± SD. The experiments were repeated three times. *p < 0.05, **p < 0.01, and ***p < 0.001.