EZH2 Mediates miR-146a-5p/HIF-1 α to Alleviate Inflammation and Glycolysis after Acute Spinal Cord Injury

Abstract

Acute spinal cord injury (ASCI) is a severe traumatic disease of the central nervous system, the underlying mechanism of which is unclear. This study was intended to study the role of EZH2 and miR-146a-5p/HIF-1α in inflammation and glycolysis after ASCI, providing reference and basis for the clinical treatment and prognosis of ASCI injury. We used lipopolysaccharide (LPS) to induce inflammation of microglia, and we constructed the ASCI animal model. qRT-PCR detected the relative expression levels of EZH2, HIF-1α, miR-146a-5p, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2 in cells and tissues. Western blot was performed to detect the expression levels of EZH2, HIF-1α, H3K27me3, IL-6, TNF-α, IL-17, PKM2, GLUT1, and HK2. ChIP verified the enrichment of H3K27me3 in the miR-146a-5p promoter region. Bioinformatics predicted the binding sites of HIF-1α and miR-146a-5p, and dual-luciferase reporter assay verified the binding of HIF-1α and miR-146a-5p. ELISA detects the levels of inflammatory factors IL-6, TNF-α, and IL-17 in the cerebrospinal fluid of rats. The GC-TOFMS was used to detect the changes of glycolytic metabolites in the cerebrospinal fluid of rats. EZH2 could mediate inflammation and glycolysis of microglia. EZH2 regulates inflammation and glycolysis through HIF-1α. EZH2 indirectly regulated the HIF-1α expression by mediating miR-146a-5p. EZH2 mediates miR-146a-5p/HIF-1α to alleviate inflammation and glycolysis in ASCI rats. In the present study, our results demonstrated that EZH2 could mediate miR-146a-5p/HIF-1α to alleviate the inflammation and glycolysis after ASCI. Therefore, EZH2/miR-146a-5p/HIF-1α might be a novel potential target for treating ASCI.

Figure 1

The inflammation, glycolysis, and HIF-1α in microglia treated with LPS were associated with upregulated EZH2. In the control group and LPS group. (a) qRT-PCR detected EZH2 expression. (b) qRT-PCR was performed to detect the expression of HIF-1α. (c) The expressions of inflammatory factors IL-6, TNF-α, and IL-17 were detected by qRT-PCR. (d) qRT-PCR detected glycolysis-related gene PKM2, GLUT1,and HK2 expressions. (e) Western blot analysis of the expression of EZH2; HIF-1α; inflammatory factors IL-6, TNF-α, and IL-17; and glycolysis-related genes PKM2, GLUT1, and HK2. ^∗P < 0.05 vs. Control group.

Figure 2

The inflammation and glycolysis in microglia were mediated by EZH2. (a, b) qRT-PCR and Western blot detected the knockdown efficiency of EZH2 in MG, respectively. ^∗P < 0.05 vs. sh-NC group. (c) Western blot was performed to detect HIF-1α; inflammatory factors IL-6, TNF-α, and IL-17; and glycolysis-related gene PKM2, GLUT1, and HK2 expressions in the Control group, sh-NC group, and sh-EZH2 group. ∗P < 0.05 vs. Control group.

Figure 3

EZH2 regulated inflammation and glycolysis through HIF-1α. (a, b) qRT-PCR and Western blot were performed to detect the overexpression of HIF-1α in microglia, respectively. ^∗P < 0.05 vs oe-NC group. (c) qRT-PCR were used to detect inflammatory factors IL-6, TNF-α, and IL-17 and glycolysis-related gene PKM2, GLUT1, and HK2 expressions. ^∗P < 0.05 vs Control group; ^#P < 0.05 vs oe-HIF-1α group. (d) Western blot detected inflammatory factors IL-6, TNF-α, and IL-17 and glycolysis-related gene PKM2, GLUT1, and HK2 expressions. ^∗P < 0.05 vs. Control group; ^#P < 0.05 vs. oe-HIF-1α group.

Figure 4

EZH2 indirectly regulated the HIF-1α expression by mediating miR-146a-5p. (a) Western blot detected the expression of H3K27me3 in the Control group and LPS group. ^∗P < 0.05 vs. Control group. (b) qRT-PCR detected miR-146a-5p expression in the Control group and LPS group. ^∗P < 0.05 vs. Control group. (c) ChIP was used to analyze the enrichment of H3K27me3 in the miR-146a-5p promoter region. ^∗P < 0.05 vs. Control group. (d) starBase predicted the binding sites of HIF-1α and miR-146a-5p, and dual-luciferase report assay verified the binding of HIF-1α and miR-146a-5p. ^∗P < 0.05 vs. WT-HIF-1α group. (e) Western blot detected the expression of HIF-1α in the NC-EZH2 group, NC-miR-146a-5p group, miR-146a-5p inhibitor group, EZH2 inhibitor+NC-miR-146a-5p group, and EZH2 inhibitor+miR-146a-5p inhibitor group. ^∗P < 0.05 vs. NC-EZH2 group; ^#P < 0.05 vs. NC-EZH2+miR-146a-5p inhibitor group; ^&P < 0.05 vs. NC-miR-146a-5p+EZH2 inhibitor. (f) Western blot detected inflammatory factors IL-6, TNF-α, and IL-17 and glycolysis-related gene PKM2, GLUT1, and HK2 expressions. (g) Inflammatory factors IL-6, TNF-α, and IL-17 and glycolysis-related gene PKM2, GLUT1, and HK2 expressions. ^∗P < 0.05 vs. NC-EZH2 group; ^#P < 0.05 vs. NC-EZH2+miR-146a-5p inhibitor group; ^&P < 0.05 vs. NC-miR-146a-5p+EZH2 inhibitor.

Figure 5

EZH2 mediates miR-146a-5p/HIF-1α to alleviate inflammation and glycolysis in ASCI rats. (a) qRT-PCR detected EZH2, miR-146a-5p, and HIF-1α expressions in the Sham group and ASCI group. (b) Western blot detected the expression of H3K27me3 in the Sham group and ASCI group. (c) qRT-PCR was performed to detect miR-146a-5p expression. (d) Western blot detected the expression of HIF-1α. (e–g) ELISA was performed to detect inflammatory factor IL-6, TNF-α, and IL-17 levels in the cerebrospinal fluid of rats. (h) Metabolomics detected the relative levels of glycolytic metabolites G6P, F6P, SUCCINATE, and MALATE. ^∗P < 0.05 vs. Sham group; ^#P < 0.05 vs. ASCI group.