Hsa_circ_0091581 promotes glioma progression by regulating RMI1 via sponging miR-1243-5p

Abstract

Glioma is a pervasive malignancy and the main cause of cancer-related deaths worldwide. Circular RNA is an important subject of cancer research, and its role and function in glioma are poorly understood. This study demonstrated that hsa_circ_0091581 is upregulated in glioma tissues and cells. The results of the CCK-8, EdU, and transwell assays indicated that hsa_circ_0091581 promotes proliferation, migration, and invasion of glioma cells. The results of the luciferase reporter and RNA immunoprecipitation assays indicated that the mechanism of the effects of hsa_circ_0091581 on glioma cells involves sponging miR-1243-5p to regulate RMI1. The results of the rescue experiments indicated that hsa_circ_0091581 regulates proliferation, migration, and invasion of glioma cells by targeting RMI1 in a miR-1243-5p dependent manner. The results of the nude mice xenograft assays showed that knockdown of hsa_circ_0091581 inhibits glioma growth in vivo. Thus, our study determined the role of hsa_circ_0091581/miR-1243-5p/RMI1 in glioma and suggests that this axis may be a novel therapeutic target in glioma.

Keywords: circ_0091581; miR-1243-5p, RMI1, glioma..

Figure 1

Hsa-circ_0091581 is upregulated in glioma tissues and cells. (A) The expression of hsa_circ_0091581 in glioma and adjacent nonneoplastic tissue (n=20). (B) The expression of hsa_circ_0091581 in glioma cells. (C-D) The RNase R assay confirmed the circular structure of hsa_circ_0091581 in U87 and LN229 cells. (E-F) Circular transcript of GPC3 (hsa_circ_0091581) was more stable than linear transcript according to the results of the actinomycin D treatment assays in U87 and LN229 cells.

Figure 2

Downregulation of hsa-circ_0091581 inhibits the proliferation, migration, and invasion of glioma cells. (A) qRT-PCR assay of the expression of hsa_circ_0091581 and GPC3 in U87 and U251 cells transfected with sh-NC or sh-circ_0091581. (B-C) Cell proliferation was assessed by CCK-8 assay after hsa_circ_0091581 downregulation in U87 and U251 cells. (D-E) Cell proliferation was assessed by EdU assay after hsa_circ_0091581 downregulation in U87 and U251 cells. (F-G) Cell migration and invasion were evaluated by transwell assay after hsa_circ_0091581 downregulation in U87 and U251 cells.

Figure 3

Hsa_circ_0091581 sponges miR-1243-5p in U87 and U251 cells. (A) The expression of miR-1243-5p in glioma and adjacent nonneoplastic tissue (n=20). (B) Pearson correlation analysis indicated a reverse correlation between hsa_circ_0091581 and miR-1243-5p. (C) The expression of miR-1243-5p in glioma cells. (D) qRT-PCR assay of the expression of miR-1243-5p in U87 and U251 cells transfected with sh-NC or sh-circ_0091581. (E) The relative luciferase activity was assayed in U87 and U251 cells cotransfected with miR-1243-5p mimics or miR-NC and hsa_circ_0091581 wild type or mutant luciferase reporter vectors. (F) Biotinylated RIP assay of the cells transfected with Bio-circ_0091581, Bio-circ_0091581-MT, or a control vector.

Figure 4

RMI1 is the functional target of miR-1243-5p in U87 and U251 cells. (A) The expression of RMI1 in glioma and adjacent nonneoplastic tissue (n=20). (B) Pearson correlation analysis indicated a reverse correlation between RMI1 and miR-1243-5p. (C) The expression of RMI1 in glioma cells. (D-E) qRT-PCR and western blot assays of the expression of RMI1 in U87 and U251 cells transfected with miR-NC or miR-1243-5p mimics. (F) The relative luciferase activity was assayed in U87 and U251 cells cotransfected with miR-1243-5p mimics or miR-NC and RMI1 wild type or mutant luciferase reporter vectors.

Figure 5

Hsa_circ_0091581 promotes the proliferation, migration, and invasion of glioma cells by targeting RMI1 in a miR-1243-5p dependent manner. (A-B) qRT-PCR and western blot assay of the expression of RMI1 in U87 and U251 cells transfected with sh-NC+miR-NC, sh-NC+miR-1243-5p mimics, miR-NC+sh-circ_0091581, or sh-circ_0091581+miR-1243-5p inhibitors. (C-D) CCK-8 assay results indicate that miR-1243-5p mimics and sh-circ_0091581 inhibit the proliferation of U87 and U251 cells, and the inhibitory effect of sh-circ_0091581 is restored by miR-1243-5p inhibitors. (E-F) EdU assay results indicate that miR-1243-5p mimics and sh-circ_0091581 inhibit the proliferation of U87 and U251 cells, and the inhibitory effect of sh-circ_0091581 is restored by miR-1243-5p inhibitors. (G-H) Transwell assay results indicate that miR-1243-5p mimics and sh-circ_0091581 inhibit the migration and invasion of U87 and U251 cells, and the inhibitory effect of sh-circ_0091581 is restored by miR-1243-5p inhibitors.

Figure 6

Hsa_circ_0091581 promotes glioma growth in vivo. (A-B) Downregulation of hsa_circ_0091581 decreases tumor growth in vivo. (C) The expression of hsa_circ_0091581 in the tumors formed in the two groups of mice. (D) Downregulation of hsa_circ_0091581 increases miR-1243-5p expression in the tumor. (E-F) Downregulation of hsa_circ_0091581 reduces RMI1 expression in the tumor.