Downregulation of hsa_circ_0001836 Induces Pyroptosis Cell Death in Glioma Cells via Epigenetically Upregulating NLRP1

Abstract

Background: It has been shown that circular RNAs (circRNAs) play a vital role in the progression of glioma. Recently, hsa_circ_0001836 was found to be upregulated in glioma tissues, but the role of hsa_circ_0001836 in glioma remains unclear.

Methods: EdU staining and flow cytometry assays were used to measure the viability and death of glioma cells. In addition, scanning electron microscopy (SEM) was used to observe the morphology of cells undergoing cell death.

Results: Hsa_circ_0001836 expression was upregulated in U251MG and SHG-44 cells. In addition, hsa_circ_0001836 knockdown significantly reduced the viability and proliferation of U251MG and SHG-44 cells. Moreover, hsa_circ_0001836 knockdown markedly induced the pyroptosis of U251MG and SHG-44 cells, evidenced by the increased expressions of NLRP1, cleaved caspase 1 and GSDMD-N. Meanwhile, methylation specific PCR (MSP) results indicated that hsa_circ_0001836 knockdown epigenetically increased NLRP1 expression via mediating DNA demethylation of NLRP1 promoter region. Furthermore, downregulation of hsa_circ_0001836 notably induced pyroptosis and inhibited tumor growth in a mouse xenograft model of glioma.

Conclusion: Collectively, hsa_circ_0001836 knockdown could induce pyroptosis cell death in glioma cells in vitro and in vivo via epigenetically upregulating NLRP1 expression. These findings suggested that hsa_circ_0001836 may serve as a potential therapeutic target for the treatment of glioma.

Keywords: NLRP1; circular RNA; glioma; methylation; pyroptosis.

Figure 1

Hsa_circ_0001836 knockdown inhibited the viability and proliferation of glioma cells. (A) Hsa_circ_0001836 levels in HA1800, U251MG, U87MG and SHG-44 cells were detected using RT-qPCR. **P < 0.01 compared with HA1800 group. (B, C) Hsa_circ_0001836 levels in U251MG and SHG-44 cells transfected with hsa_circ_0001836 siRNA1, hsa_circ_0001836 siRNA2 or hsa_circ_0001836 siRNA3 analyzed by RT-qPCR. (D) Cell viability analyzed by CCK-8 assay in U251MG and SHG-44 cells transfected with hsa_circ_0001836 siRNA3 for 72 h. (E, F) EdU staining assay was used to determine cell proliferation. **P < 0.01 vs. siRNA-ctrl group.

Figure 2

Downregulation of hsa_circ_0001836 induced pyroptosis cell death in glioma cells. U251MG and SHG-44 cells were transfected with hsa_circ_0001836 siRNA3 for 72 h. (A–D) Flow cytometry was used to determine cell death. (E, F) Dying cells revealed by SEM. **P < 0.01 vs. siRNA-ctrl group.

Figure 3

Downregulation of hsa_circ_0001836 induced pyroptosis in glioma cells via activation of NLRP1-GSDMD signaling. U251MG cells were transfected with hsa_circ_0001836 siRNA3 for 72 h. (A–D) Western blot analysis of NLRP1, cleaved caspase 1, GSDMD-N levels in U251MG cells. The relative expressions of NLRP1, cleaved caspase 1, GSDMD-N in U251MG cells normalized to β-actin. (E) The level of IL-1β in the culture supernatants of U251MG cells was measured using ELISA. (F) The level of IL-18 in the culture supernatants of U251MG cells was measured using ELISA. **P < 0.01 vs. siRNA-ctrl group.

Figure 4

Downregulation of hsa_circ_0001836 induced pyroptosis in glioma cells via epigenetically upregulating NLRP1. (A) U251MG cells were transfected with hsa_circ_0001836 siRNA3 for 72 h. DNA methylation of the NLRP1 promoter region was analyzed by MSP. “U” and “M” represents unmethylated and methylated signal intensity, respectively. The product length of the methylated primers of NLRP1 is 125 bp; and the product length of the unmethylated primers of NLRP1 is 127 bp. (B) The level of NLRP1 in U251MG cells transfected with NLRP1 siRNA1, NLRP1 siRNA2 or NLRP3 siRNA3 analyzed by RT-qPCR. (C) U251MG cells were transfected with hsa_circ_0001836 siRNA3 or co-transfected with hsa_circ_0001836 siRNA3 and NLRP1 siRNA2 for 72 h. CCK-8 assay was applied to determine cell viability. (D) Cell death was detected by flow cytometry. (E) Dying cells revealed by SEM. (F, G) Western blot analysis of NLRP1, GSDMD-N levels in U251MG cells. The relative expressions of NLRP1, GSDMD-N in U251MG cells normalized to β-actin. *P < 0.05, **P < 0.01 vs. siRNA-ctrl group; ^##P < 0.01 vs. hsa_circ_0001836 siRNA3 group.

Figure 5

Knockdown of hsa_circ_0001836 inhibited tumorigenesis in U251MG xenografts in vivo. (A) Xenograft tumor volume were measured weekly. (B, C) Xenografts tumors were imaged on day 21, and the tumor weights were calculated. (D) The level of hsa_circ_0001836 in tumor tissues was analyzed by RT-qPCR. **P < 0.01 vs. control group.

Figure 6

Knockdown of hsa_circ_0001836 inhibited tumorigenesis in U251MG xenografts in vivo via activation of NLRP1-GSDMD signaling. U251 cells (1 × 10⁷ cells) were injected into nude mice subcutaneously, and hsa_circ_0001836 siRNA3 (50 nM) was injected into the tumors directly twice a week. (A–D) Western blot analysis of NLRP1, cleaved caspase 1, GSDMD-N levels in tumor tissues. The relative expressions of NLRP1, cleaved caspase 1, GSDMD-N in tumor tissues normalized to β-actin. (E, F) IHC analysis was applied to determine the expressions of IL-1β and IL-18 in tumor tissues. **P < 0.01 vs. control group.