Circular RNA circ_0026359 Enhances Cisplatin Resistance in Gastric Cancer via Targeting miR-1200/POLD4 Pathway

Abstract

Human gastric cancer is one of the most common malignant tumors with a poor prognosis. Cisplatin (CDDP) is a well-known first-line chemotherapeutic drug. Acquired resistance retards the clinical application of CDDP in gastric cancer. In this study, circular RNA circ_0026359 was demonstrated to be overexpressed in gastric cancer tissues/cells compared with normal gastric tissues/cells and was overexpressed in CDDP-resistant gastric cancer tissues/cells compared with CDDP-sensitive gastric cancer tissues/cells. High levels of circ_0026359 were associated with low overall survival (OS) and relapse-free survival (RFS) rates in gastric cancer patients. circ_0026359 was examined to promote CDDP resistance in gastric cancer cells. circ_0026359 directly interacted and negatively regulated miR-1200. POLD4 was a direct target of miR-1200. miR-1200/POLD4 pathway mediated the promoting role of circ_0026359 in CDDP resistance of gastric cancer. circ_0026359 could be used as a potential target for CDDP-resistant gastric cancer therapy.

Figure 1

circ_0026359 was overexpressed in gastric cancer and associated with CDDP resistance and poor survival rates in gastric cancer patients. (a) Expression levels of circ_0026359 in 55 pairs of gastric cancer tissues and normal gastric tissues were examined by RT-qPCR. (b) Expression levels of circ_0026359 in 33 CDDP-sensitive (CS) and 22 CDDP-resistant (CT) gastric cancer tissues. (c) Expression levels of circ_0026359 in normal gastric cell GES-1 and gastric cancer cells HGC-27, AGS, BGC-823, SGC-7901, and MKN-45 were examined by RT-qPCR. (d) Expression levels of circ_0026359 in CDDP-resistant gastric cancer cells SGC-7901/CDDP and MKN-45/CDDP and parental gastric cells SGC-7901 and MKN-45. GAPDH was examined as control for RT-qPCR. (e) Overall survival (OS) rates and (f) relapse-free survival (RFS) rates in gastric cancer patients with high circ_0026359 expression and low circ_0026359 expression were analyzed by Kaplan-Meier curves. ^∗∗P < 0.01; ^∗∗∗P < 0.001.

Figure 2

Depletion of circ_0026359 decreased CDDP resistance of gastric cancer cells. CDDP-resistant gastric cancer cells SGC-7901/CDDP and MKN-45/CDDP were transfected with si-circ_0026359#1, si-circ_0026359#2, or siNC. (a) Expression levels of circ_0026359 were examined by RT-qPCR. GAPDH was used as control. (b, c) These cells were treated with 1 μg/ml CDDP for 24, 36, and 48 hours, and cell viabilities were examined by the MTT assay. (d) IC50 for CDDP in these cells were analyzed by the MTT assay. (e) Cell colony formation assay was performed in these cells with 1 μg/ml CDDP treated for 48 hours. (f) Caspase-3/7 assay, (g) DNA fragmentation assay, and (h) flow cytometry with Annexin V-FITC and PI staining were performed in these cells with 1 μg/ml CDDP treated for 48 hours. ^∗∗P < 0.01.

Figure 3

miR-1200 directly bound to circ_0026359. (a, b) Location of circ_0026359 in SGC-7901/CDDP and MKN-45/CDDP cells was examined by nuclear and cytoplasmic separation assay. circ_0026359 was detected by RT-qPCR. U6 and GAPDH were examined as control. (c) Predicted consequential pairing of circ_0026359 with miR-1200 and the mutant consequence of circ_0026359 used in luciferase reporter assay. (d, e) Luciferase reporter assay of SGC-7901/CDDP and MKN-45/CDDP cells cotransfected with miR-1200 mimics/negative control miRNA (miR-NC) and luciferase reporter plasmid pmirGLO containing wild-type circ_0026359 sequence (hsa_circ_0026359-wt)/mutant circ_0026359 sequence (hsa_circ_0026359-mt). (f) RT-qPCR was carried out to examine miR-1200 levels in SGC-7901/CDDP and MKN-45/CDDP cells after transfection with si-circ_0026359#1, si-circ_0026359#2, or siNC. U6 was detected as control. (g, h) RNA pull-down assay: (g) circ_0026359 and (h) miR-1200 were enriched by biotinylated circ_0026359 probe, and their levels were examined by RT-qPCR. ^∗P < 0.05; ^∗∗P < 0.01. ns: no significance.

Figure 4

POLD4 was a direct target of miR-1200. (a) Predicted consequential pairing of miR-1200 with the POLD4 3′UTR and the mutant consequence of POLD4 3′UTR used in the luciferase reporter assay. (b, c) Luciferase reporter assay of SGC-7901/CDDP and MKN-45/CDDP cells cotransfected with miR-1200/miR-NC mimics and luciferase reporter plasmid pmirGLO containing wild-type POLD4 3′UTR sequence (hsa_POLD4 3′UTR-wt)/mutant POLD4 3′UTR sequence (hsa_POLD4 3′UTR-mt). (d) mRNA levels of POLD4 in SGC-7901/CDDP and MKN-45/CDDP cells after transfection with miR-1200/miR-NC mimics were examined by RT-qPCR. U6 was detected as control. (e) Protein levels of POLD4 in SGC-7901/CDDP and MKN-45/CDDP cells after transfection with miR-1200/miR-NC mimics were examined by western blot. α-Tubulin was detected as control. ^∗P < 0.05; ^∗∗P < 0.01. ns: no significance.

Figure 5

miR-1200 rescued the decreased CDDP resistance due to circ_0026359 depletion in CDDP-resistant gastric cancer cells. SGC-7901/CDDP cells were cotransfected with si-circ_0026359#1/si-NC and miR-1200 inhibitor/NC-inhibitor. (a) Expression levels of miR-1200 and POLD4 mRNA were examined by RT-qPCR. U6 and GAPDH were detected as control, respectively. (b) Protein levels of POLD4 were examined by western blot. α-Tubulin was detected as control. (c) MTT assay (1 μg/ml CDDP treated for 24, 36, and 48 hours), (d) IC50 analysis for CDDP, (e) cell colony formation assay (1 μg/ml CDDP treated for 48 hours), (f) caspase-3/7 assay (1 μg/ml CDDP treated for 48 hours), (g) DNA fragmentation assay (1 μg/ml CDDP treated for 48 hours), and (h) flow cytometry with Annexin V-FITC and PI staining (1 μg/ml CDDP treated for 48 hours) were performed, respectively, in SGC-7901/CDDP cells after cotransfection. ^∗∗P < 0.01.