A Novel Circular RNA Mediates Pyroptosis of Diabetic Cardiomyopathy by Functioning as a Competing Endogenous RNA

Abstract

Diabetic cardiomyopathy (DCM) is a vital cause of fatalities in diabetic patients. The programmed death of cardiomyocytes and inflammation critically contribute to cardiac hypertrophy and fibrosis in DCM. Furthermore, circular RNA (circRNA) is a key regulator of various diseases. However, the role of circRNAs in DCM remains to be elucidated. Our previous study found that pyroptosis was markedly activated in the cardiomyocytes subjected to high-glucose conditions, and miR-214-3p regulated the expression of caspase-1. The aim of this study was to elucidate whether circRNA is involved in DCM pyroptosis via the miR-214-3p/caspase-1 pathway. Herein, we identified that hsa_circ_0076631, named caspase-1-associated circRNA (CACR), was increased both in high-glucose-treated cardiomyocytes and in the serum of diabetic patients. CACR also sponged an endogenous miR-214-3p to sequester and inhibit its expression. CACR knockdown in cardiomyocytes counteracted high-glucose-induced caspase-1 activation. Conversely, miR-214-3p knockdown partially abolished the beneficial effects of CACR silencing on pyroptosis in cardiomyocytes. Therefore, this study elucidated that CACR might be a novel therapeutic target via the CACR/miR-214-3p/caspase-1 pathway in DCM.

Keywords: caspase-1; circular RNA; diabetic cardiomyopathy; miR-214-3p; pyroptosis.

Figure 1

miR-214-3p Inhibited Pyroptosis Activation in High-Glucose-Treated AC16 Cells (A) qRT-PCR was conducted to detect the expression levels of NLRP3, caspase-1, and IL-1β in AC16 cells. (B–D) Western blots were conducted to detect the protein expression levels of NLRP3 (B), caspase-1 (C), and IL-1β(Δ). GAPDH was detected as the internal control. (E) qRT-PCR was conducted to detect the expression levels of miR-214-3p in AC16 cells. *p < 0.05 versus the control group. (F) High-glucose-treated AC16 cells were transfected with NC, miR-214-3p mimics, AMO-NC, and AMO-214-3p, and miR-214-3p expression levels were detected by qRT-PCR. (G) The relative mRNA expression levels of caspase-1 were detected. (H) Western blots were performed to detect the protein expression of caspase-1. n = 3. *p < 0.05 versus the HG+NC group, #p < 0.05 versus the HG+AMO-NC group.

Figure 2

CACR Silencing Regulates Caspase-1 and miR-214-3p in Cardiomyocytes (A) qRT-PCR was used to detect six candidate circRNAs that have potential binding sites with miR-214-3p in AC16 cells. n = 3. (B) CACR expression levels in the serum of non-diabetic patients and diabetic patients. n = 10. (C) High-glucose-treated AC16 cells were transfected with ASO-NC and ASO-CACR. The relative expression levels of CACR, miR-214-3p, and caspase-1 were detected by qRT-PCR. (D) Relative caspase-1 protein expression was detected in AC16 cells. (E) FISH assays were performed to detect the location of CACR in AC16 cells. CACR, 18 s, U6, red; nuclei, blue. Scale bar, 40 μm. (F) CACR contains 11 potential binding sites complementary to miR-214-3p as analyzed by bioinformatics programs. (G) SLC29A1 is the corresponding linear sequence of CACR. The binding sequence of SLC29A1 and miR-214-3p and the mutant sequence of SLC29A1 are shown. (H) Wide-type (WT) and mutant type (MUT) SLC29A1 were cloned downstream of the luciferase vector and co-transfected with miR-NC and miR-214-3p mimics into HEK293 cells. Luciferase activity was detected using a luciferase assay. The relative Rluc/Luc ratio is shown. n = 3. *p < 0.05.

Figure 3

CACR Regulates Caspase-1 Expression via Sponging miR-214-3p in Cardiomyocytes (A) Immunofluorescence analysis was performed to detect the expression level of caspase-1. A representative image is shown in the left panel. Caspase-1, red; nuclei, blue. Scale bar, 60 μm. The calculated fluorescence intensity is shown in the right panel. (B) The relative mRNA expression levels of caspase-1 were detected by qRT-PCR. (C) Western blots were performed to detect the protein expression of caspase-1. n = 3. *p < 0.05 compared with the ASO-NC+AMO-NC group, #p < 0.05 compared with the HG+ASO-NC+AMO-NC group, &p < 0.05 compared with the HG+ASO-CACR+AMO-NC group.

Figure 4

CACR Regulates Pyroptosis via the miR-214-3p/Caspase-1 Pathway in AC16 Cells (A) Immunofluorescence analysis was performed to detect the expression levels of NLRP3. A representative image is shown in the left panel. NLRP3, red; DAPI, blue. Scale bar, 60 μm. The calculated fluorescence intensity is shown in the right panel. (B) The relative expression levels of NLRP3 were detected by qRT-PCR. (C) Immunofluorescence analysis was performed to detect the expression levels of IL-1β. (D) The relative expression levels of IL-1β were detected by qRT-PCR. n = 3. *p < 0.05 compared with the ASO-NC+AMO-NC group, #p < 0.05 compared with the HG+ASO-NC+AMO-NC group, &p < 0.05 compared with the HG+ASO-CACR+AMO-NC group.

Figure 5

The CACR/miR-214-3p Pathway Regulates DNA Fracture in AC16 Cells (A) A representative image of the TUNEL assay is shown. TUNEL-positive cells, green; nuclei, blue. Scale bar, 60 μm. (B) TUNEL-positive cells were analyzed. n = 3. *p < 0.05 compared with the ASO-NC+AMO-NC group, #p < 0.05 compared with the HG+ASO-NC+AMO-NC group, &p < 0.05 compared with the HG+ASO-CACR+AMO-NC group.

Figure 6

Proposed Model of the Function of CACR in Regulating Pyroptosis via miR-214-3p in High-Glucose-Treated Cardiomyocytes