Silencing circPalm2 inhibits sepsis-induced acute lung injury by sponging miR-376b-3p and targeting MAP3K1

Abstract

The apoptosis and inflammation of pulmonary epithelial cells are important pathogenic factors of sepsis-induced acute lung injury (ALI). Upregulation of circPalm2 (circ_0001212) expression levels has been previously detected in the lung tissue of ALI rats. Herein, the biological significance and detailed mechanism of circPalm2 in ALI pathogenesis were investigated. In vivo models of sepsis-induced ALI were established by treating C57BL/6 mice with cecal ligation and puncture (CLP) surgery. Murine pulmonary epithelial cells (MLE-12 cells) were stimulated with lipopolysaccharide (LPS) to establish in vitro septic ALI models. MLE-12 cell viability and apoptosis were evaluated by CCK-8 assay and flow cytometry analysis, respectively. The pathological alterations of the lung tissue were analysed based on hematoxylin-eosin (H&E) staining. Cell apoptosis in the lung tissue samples was examined by TUNEL staining assay. LPS administration suppressed the viability and accelerated the inflammation and apoptotic behaviours of MLE-12 cells. CircPalm2 displayed high expression in LPS-stimulated MLE-12 cells and possessed circular characteristics. The silencing of circPalm2 impeded apoptosis and inflammation in LPS-stimulated MLE-12 cells. Mechanistically, circPalm2 bound with miR-376b-3p, which targeted MAP3K1. In rescue assays, MAP3K1 enhancement reversed the repressive effects of circPalm2 depletion on LPS-triggered inflammatory injury and MLE-12 cell apoptosis. Furthermore, the lung tissue collected from CLP model mice displayed low miR-376b-3p expression and high levels of circPalm2 and MAP3K1. CircPalm2 positively regulated MAP3K1 expression by downregulating miR-376b-3p in murine lung tissues. Importantly, circPalm2 knockdown attenuated CLP-induced inflammation, apoptosis, and pathological alterations in lung tissues collected from mice. Silenced circPalm2 inhibits LPS-induced pulmonary epithelial cell dysfunction and mitigates abnormalities in lung tissues collected from CLP-stimulated mice via the miR-376b-3p/MAP3K1 axis in septic ALI.

Supplementary information: The online version contains supplementary material available at 10.1007/s43188-022-00169-7.

Keywords: Acute lung injury; Inflammation; MAP3K1; Sepsis; circPalm2; miR-376b-3p.

Fig. 1

LPS induces MLE-12 cell apoptosis and inflammation. (A) CCK-8 assay for evaluating MLE-12 cell viability in the Con and LPS groups. (B) ELISA for determining proinflammatory cytokine concentrations in MLE-12 cells in the Con and LPS groups. (C, D) Flow cytometry analysis for determining MLE-12 cell cycle transition and cell apoptosis in the Con and LPS groups. (E) Western blotting for determining apoptosis-related marker protein levels in MLE-12 cells of the Con and LPS groups. **p < 0.01, ***p < 0.001

Fig. 2

Characterization of circPalm2. (A) Schematic diagram of circPalm2 formed by back-splicing from the Palm2 premRNA at the chromosome, as shown by the circBase database (http://www.circbase.org/). (B) The levels of circPalm2 and linear Palm2 were measured after treatment with RNase R. (C) CircPalm2 and linear Palm2 levels were estimated after actinomycin D treatment. (D) RT‒qPCR for evaluating circPalm2 expression in MLE-12 cells of the Con and LPS groups. (E) Nuclear-cytoplasmic fractionation assay for determining the subcellular localization of circPalm2 in MLE-12 cells. **p < 0.01, ***p < 0.001

Fig. 3

CircPalm2 depletion ameliorates LPS-evoked MLE-12 cell damage. (A) RT‒qPCR for transfection efficiency of sh-circPalm2. (B) CCK-8 assay for evaluating LPS-treated MLE-12 cell viability after downregulating circPalm2. (C) ELISA for determining the proinflammatory cytokine concentration in LPS-stimulated MLE-12 cells after downregulating circPalm2. (D, E) Flow cytometry analysis for determining LPS-treated MLE-12 cell cycle transition and cell apoptosis after downregulating circPalm2. (F) Western blotting for determining apoptosis-related marker protein levels in LPS-stimulated MLE-12 cells after downregulating circPalm2. **p < 0.01

Fig. 4

CircPalm2 sponges miR-376b-3p. (A) Potential miRNAs (mmu-miR-330-5p, mmu-miR-326-3p, mmu-miR-3077-3p, mmu-miR-7661-3p and mmu-miR-376b-3p) that can interact with circPalm2 were searched on the ENCORI website. (B) RT‒qPCR for detecting the impact of LPS on miRNAs in MLE-12 cells. (C) miR-330-5p and miR-376b-3p expression in response to circPalm2 silencing was measured by qPCR in MLE-12 cells. (D) RT‒qPCR for detecting the impact of miR-376b-3p mimics on the levels of miR-376b-3p and circPalm2 in MLE-12 cells. (E) Bioinformatics analysis of the possible binding area between circPalm2 and miR-376b-3p. (F) Luciferase reporter assays were performed to verify the binding relationship between circPalm2 and miR-376b-3p. **p < 0.01, ***p < 0.001

Fig. 5

MiR-376b-3p targets MAP3K1. (A) RT‒qPCR for detecting the impact of LPS on the mRNA expression of 3 candidate genes (MAP3K1, Scd2 and Hnrnpa0) in MLE-12 cells. (B, C) Western blotting for detecting the impact of LPS on MAP3K1 protein expression in MLE-12 cells. (D) According to the prediction from the ENCORI website, a binding site for miR-376b-3p is present in the MAP3K1 3’UTR. (E) Luciferase reporter assays were conducted to confirm the interaction between miR-376b-3p and the MAP3K1 3’UTR. (F, H, I) The protein level of MAP3K1 in the context of miR-376b-3p upregulation, miR-376b-3p inhibition, or circPalm2 silencing was measured by western blotting. (G) The knockdown efficacy of the miR-376b-3p inhibitor in MLE-12 cells was evaluated by RT‒qPCR. **p < 0.01, ***p < 0.001

Fig. 6

miR-376b-3p overexpression attenuates LPS-induced cell apoptosis and the inflammatory response, and the alterations were counteracted by overexpression of MAP3K1. (A, B) RT‒qPCR and western blotting for evaluating the transfection efficiency of pcDNA3.1/MAP3K1. (C) CCK-8 assay for detecting cell viability after LPS treatment and plasmid (miR-NC, miR-376b-3p mimics, or miR-376b-3p mimics + pcDNA3.1/MAP3K1) transfection. (D) ELISA for measuring the concentrations of inflammatory cytokines in cells with the above treatment. (E, F) Flow cytometry analysis for the detection of cell cycle progression and cell apoptosis. (G) Protein levels of apoptosis-related genes were quantified using western blotting. **p < 0.01, ***p < 0.001

Fig. 7

MAP3K1 enhancement reverses the impact of circPalm2 knockdown on LPS-induced MLE-12 cell apoptosis and inflammation. (A) CCK-8 assay for evaluating LPS-treated MLE-12 cell viability after downregulation of circPalm2 alone or simultaneous downregulation of circPalm2 and overexpression of MAP3K1. (B) ELISA for determining the proinflammatory cytokine concentrations in LPS-stimulated MLE-12 cells after downregulation of circPalm2 alone or simultaneous downregulation of circPalm2 and overexpression of MAP3K1. (C–E) Flow cytometry analysis for determining LPS-treated MLE-12 cell cycle transition and apoptosis after downregulation of circPalm2 alone or simultaneous downregulation of circPalm2 and overexpression of MAP3K1. (F) Western blotting for determining apoptosis-related marker protein levels in LPS-stimulated MLE-12 cells after downregulation of circPalm2 alone or simultaneous downregulation of circPalm2 and overexpression of MAP3K1. *p < 0.05, **p < 0.01

Fig. 8

The expression of circPalm2, miR-376b-3p and MAP3K1 in the lung tissues of CLP-induced ALI mice. (A–C) RT‒qPCR for evaluating circPalm2, miR-376b-3p and MAP3K1 levels in murine lung tissues from the sham, CLP, CLP + Ad-NC and CLP + Ad-Sh-circPalm2 groups. (D–F) Pearson correlation analysis of the correlations among circPalm2 expression, miR-376b-3p expression and MAP3K1 expression in the lung tissues of CLP-induced ALI mice (n = 24). (G, H) miR-330-5p and ROCK2 expression levels in lung tissues collected from the four experimental groups were detected by PCR. (I–K) Expression associations among circPalm2, miR-330-5p, and ROCK2 in lung tissues of CLP-treated ALI mice (n = 24) by Pearson correlation analyses. *p < 0.05, **p < 0.01

Fig. 9

Knockdown of circPalm2 inhibits lung injury in CLP-treated mice. (A) H&E staining to observe the histopathological characteristics of lung tissues from the sham, CLP, CLP + Ad-NC and CLP + Ad-Sh-circPalm2 groups (scale bar = 50 μm). (B) Intensity of the injury score of each group. (C) Body weight of each group. (D–F) ELISA for determining the proinflammatory cytokine secretion in each group. (G) Western blotting was performed to evaluate the protein levels of apoptosis-related genes (Bax and Bcl-2) in the lung tissue of the four experimental groups. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 10

Silencing circPalm2 inhibits JNK1/2 signalling by downregulating MAP3K1 via miR-376b-3p. Western blotting was performed to quantify phosphorylated JNK1/2 (p-JNK1/2) levels (A) in MLE-12 cells of the control group, LPS group, and LPS + MAP3K1 group; (B) in LPS-induced MLE-12 cells of the miR-NC, miR-376b-3p, and miR-376b-3p + MAP3K1 groups; (C) in LPS-stimulated MLE-12 cells of the sh-NC, sh-circPalm2, and sh-circPalm2 + MAP3K1 groups. (D) MAP3K1 and p-JNK1/2 levels in lung tissues of the sham group, CLP group, CLP + Ad-NC, and CLP + Ad-Sh-circPalm2 group were quantitated by western blotting. **p < 0.01, ***p < 0.001

Fig. 11

Inhibition of JNK1/2 signalling ameliorates LPS-induced cell injury and inflammation. After cells were treated with LPS or JNK-in-8, (A) a CCK-8 assay was conducted to detect cell viability; (B) ELISA was performed to measure the accumulation of inflammatory factors; (C, D) flow cytometry was utilized for cell cycle and apoptosis detection; and (E) western blotting was performed to quantify the protein levels of the apoptosis-related genes (Bax and Bcl-2). **p < 0.01, ***p < 0.001