Silencing circ_0043256 inhibited CoCl2-induced proliferation, migration, and aerobic glycolysis in gastric cancer cells

Abstract

We aimed to explore the role of circular RNA 0043256 (circ_0043256) in gastric cancer (GC) and its underlying mechanisms. The impact of circ_0043256 silencing on the proliferation, migration, apoptosis, and aerobic glycolysis of MKN-45 and AGS cells induced by CoCl2 was assessed through the utilization of CCK-8, wound healing assay, flow cytometry, and metabolic analysis. The interaction between circ_0043256 and miR-593-5p, as well as the involvement of the miR-593-5p/RRM2 axis in gastric cancer, were confirmed via luciferase assay, Western blot, and bioinformatics analysis. We found that circ_0043256 was up-regulated in GC tissues and CoCl2-treated MKN-45 and AGS cells. Silencing of circ_0043256 reversed CoCl2-induced proliferation, migration, and aerobic glycolysis in MKN-45 and AGS cells. Additionally, circ_0043256 silencing enhanced cell apoptosis and G2/M phase cell cycle arrest in response to CoCl2 treatment. Furthermore, the miR-593-5p/RRM2 axis was identified as a regulatory mechanism for circ_0043256 function in GC. Silencing of circ_0043256 and miR-593-5p mimic co-transfection significantly inhibited CoCl2-induced cellular responses in MKN-45 and AGS cells. A glycolysis inhibitor 2-DG further enhanced the inhibitory effect of circ_0043256 silencing on aerobic glycolysis of CoCl2-induced MKN-45 and AGS cells. Additionally, the inhibition of circ_0043256 resulted in a reduction in tumor volume and the expression of proliferation marker proteins in nude mice. Moreover, the suppression of circ_0043256 led to an increase in miR-593-5p expression and a decrease in RRM2 expression, ultimately causing a decrease in glycolytic-related proteins associated with the glycolytic pathway. Targeting this axis may offer a novel therapeutic approach for treating GC.

Keywords: Aerobic glycolysis; Circ_0043256; Gastric cancer; RRM2; miR-593-5p.

Fig. 1

Circ_0043256 was up-regulated in GC tissues and CoCl2-induced MKN-45 and AGS cells. (A) Expression of hsa_circ_0043256 in GC tissues and normal tissues showed by GSE78092 database. (B) The expression of the top 25 up-regulated circ_RNAs in tumor tissues and adjacent non-tumor tissues was assayed by RT-qPCR analysis. Numbers indicate mean. (C) The expression of circ_0043256 in tumor tissues and adjacent non-tumor tissues was measured by RT-qPCR analysis. ^##P < 0.01 (vs. Control). (D) The expression of circ_0043256 in the control human gastric mucosal epithelial cells GES-1, the human GC cell lines MKN-45, AGS, SNU-16, SNU-216, HGC-27, and MKN-7 (#CL-0574) was tested by RT-qPCR analysis. ^*P < 0.05, ^**P < 0.01 (vs. GES-1). (C) The expression of circ_0043256 was measured by RT-qPCR analysis.^**P < 0.01 (vs. siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC).

Fig. 2

Circ_0043256 regulated CoCl2-induced proliferation, migration, and apoptosis of MKN-45 and AGS cells. MKN-45 and AGS cells were exposed to different concentrations (50 µM, 100 µM, 200 µM, and 300 µM) of CoCl2 to induce a hypoxic environment. (A–C) Western blot analysis was performed to detect the expression levels of glycolytic related proteins HK2 and GLUT1 in MKN-45 and AGS cells induced by different concentrations of CoCl2. β-actin is a loading control. (D) The expression of si-circ_0043256 was quantified using RT-qPCR analysis. ^*P < 0.05 (vs. 0 µM), ^**P < 0.01 (vs. 0 µM). (E) MKN-45 and AGS cells were transfected with si-circ_0043256 and treated with CoCl2. The expression level of si-circ_0043256 was determined through RT-qPCR analysis (F and G) The proliferation of MKN-45 and AGS cells were evaluated by CCK-8 assay. (H–J) MKN-45 and AGS cell migration was tested by Wound-healing assay. Experiments were terminated after scratching for 48 h. (K-M) Flow cytometry was used to detect apoptosis and measure the rate of apoptosis. (N-P) The cell cycle of MKN-45 and AGS cells was examined by flow cytometry. ^*P < 0.05 (vs. siRNA-NC), ^**P < 0.01 (vs. siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC).

Fig. 3

Circ_0043256 regulated CoCl2-induced aerobic glycolysis of MKN-45 and AGS cells. MKN-45 and AGS cells were transfected with si-circ_0043256 and treated with CoCl2. (A and B) The ATP production, glucose consumption, and lactic acid content in CoCl2-induced MKN-45 and AGS cells were detected by commercial kits. (C and D) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 in AGS cells were determined via Western Blot analysis. β-actin is a loading control. (E and F) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 in MKN-45 cells were measured by Western Blot analysis. β-actin is a loading control. ^*P < 0.05 (vs. siRNA-NC), ^**P < 0.01 (vs. siRNA-NC), ^#P < 0.05 (vs. CoCl2 + siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC).

Fig. 4

Circ_0043256 targeted miR-593-5p/RRM2axis. (A) The targeting relationship between circ_0043256 and miR-593-5p was predicted using circMine database (http://www.biomedical-web.com/circmine/circRNA-miRNA_prediction). Diagram of the predicted miR-593-5p binding site in the 3’UTR sequences of circ_0043256. (C) The luciferase activity in 293T cells transfected with miR-593-5p mimic or normal control (NC) mimic. ^**P < 0.01 (vs. NC mimic). (D) The expression of miR-593-5p was measured by RT-qPCR analysis. (E) miRNA target prediction software (TargetScanHuman 8.0, https://www.targetscan.org/) was used to identify potential miR-593-5p target genes. (F) The luciferase activity in 293T cells transfected with miR-593-5p mimic or NC mimic. ^**P < 0.01 (vs. NC mimic). (G) The mRNA expression of RRM2 was measured by RT-qPCR analysis. (H) The protein levels of RRM2 were measured by Western Blot analysis. β-actin is a loading control. ^*P < 0.05 (vs. Con), ^**P < 0.01 (vs. Con),^#P < 0.05 (vs. CoCl2 + siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC).

Fig. 5

Circ_0043256 regulated CoCl2-induced proliferation, migration, and apoptosis of MKN-45 and AGS cells by targeting the miR-593-5p/RRM2 axis. MKN-45 and AGS cells were transfected with si-circ_0043256 and/or miR-593-5p mimic to investigate their effects on CoCl2-induced cellular responses. (A) The expression of miR-593-5p was quantified using RT-qPCR analysis. (B and C) Cell proliferation in MKN-45 and AGS cells was assessed by the CCK-8 assay. (D-F) Wound-healing assay was performed to evaluate cell migration in MKN-45 and AGS cells. Experiments were terminated 48 h after scratching. (G-I) Flow cytometry was employed to detect apoptosis and determine the apoptotic rate. (J-L) Cell cycle analysis of MKN-45 and AGS cells was conducted using flow cytometry. ^**P < 0.01 (vs. siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC), ^&P < 0.05 (vs. CoCl2 + si-circ_0043256 + mimic-NC), ^&&P < 0.01 (vs. CoCl2 + si-circ_0043256 + mimic-NC).

Fig. 6

Circ_0043256 regulated CoCl2-induced aerobic glycolysis of MKN-45 and AGS cells by targeting the miR-593-5p/RRM2 axis. MKN-45 and AGS cells were transfected with si-circ_0043256 and/or miR-593-5p mimic to investigate their effects on CoCl2-induced aerobic glycolysis. (A and B) The ATP production, glucose consumption, and lactic acid content in CoCl2-induced MKN-45 and AGS cells were assessed using commercially available kits. (C and D) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 in AGS cells were determined by Western Blot analysis with β-actin serving as a loading control. (E and F) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 in MKN-45 cells were measured via Western Blot analysis. β-actin is a loading control. ^**P < 0.01 (vs. siRNA-NC), ^#P < 0.05 (vs. CoCl2 + siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC), ^&P < 0.05 (vs. CoCl2 + si-circ_0043256 + mimic-NC), ^&&P < 0.01 (vs. CoCl2 + si-circ_0043256 + mimic-NC).

Fig. 7

2-DG further enhanced the inhibitory effect of circ_0043256 silencing on cell progression of CoCl2-induced MKN-45 and AGS cell. The MKN-45 and AGS cells were transfected with si-circ_0043256 and treated with 2-DG. (A and B) Cell proliferation in MKN-45 and AGS cells was assessed by the CCK-8 assay. (C–E) Wound-healing assay was performed to evaluate cell migration in MKN-45 and AGS cells. Experiments were terminated 48 h after scratching. (F–H) Flow cytometry was employed to detect apoptosis and determine the apoptotic rate. (I–K) Cell cycle analysis of MKN-45 and AGS cells was tested using flow cytometry. ^**P < 0.01 (vs. siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC), ^&P < 0.05 (vs. CoCl2 + 2-DG + siRNA-NC), ^&&P < 0.01 (vs. CoCl2 + 2-DG + siRNA-NC).

Fig. 8

2-DG further enhanced the inhibitory effect of circ_0043256 silencing on aerobic glycolysis of CoCl2-induced MKN-45 and AGS cells. The MKN-45 and AGS cells were transfected with si-circ_0043256 and treated with 2-DG. (A and B) The ATP production, glucose consumption, and lactic acid content in CoCl2-induced MKN-45 and AGS cells were assessed using commercially available kits. (C and D) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 in AGS cells were determined by Western Blot analysis with β-actin serving as a loading control. (E and F) The protein levels of HIF-1α, HK2, ENO1, LDHA, GLUT1,and PKM2 in MKN-45 cells were measured via Western Blot analysis. β-actin is a loading control. ^*P < 0.05 (vs. siRNA-NC), ^**P < 0.01 (vs. siRNA-NC), ^##P < 0.01 (vs. CoCl2 + siRNA-NC), ^&&P < 0.01 (vs. CoCl2 + 2-DG + siRNA-NC).

Fig. 9

The silencing of circ_0043256 inhibited tumor growth and glycolysis through the miR-593-5p/RRM2 axis. (A and B) Compared with the control group, circ_0043256 silencing inhibited the tumor weight. (C and D) Western blot was used to assay determined Ki67 and PCNA. β-actin is a loading control. (E) The expression of circ_0043256 was performed through RT-qPCR analysis. (F) The expression of miR-593-5p was quantified using RT-qPCR analysis. (G and H) IHC stain of Ki67 and PCNA, magnification is 400×. (I–K) Western blot was used to assay determined HIF-1α, HK2, ENO1, LDHA, GLUT1, and PKM2 expression in tumor tissues. ^**P < 0.05 (vs. Normoxic), ^#P < 0.05 (vs. AAV-shRNA-NC), ^##P < 0.01 (vs. AAV-shRNA-NC).