CircRNA_0044556 diminishes the sensitivity of triple‑negative breast cancer cells to adriamycin by sponging miR‑145 and regulating NRAS

Abstract

CircRNAs are associated with adriamycin (ADM) resistance in triple‑negative breast cancer (TNBC), but the mechanism is unknown. Reverse transcription‑quantitative PCR was applied to quantify circular RNA (circRNA)_0044556, microRNA (miR)‑145 and NRAS proto‑oncogene, GTPase (NRAS) in TNBC tissues and cells with or without ADM treatment. Following ADM treatment, the effects of circRNA_0044556 on the viability, ADM resistance, apoptosis and migration of TNBC cells were investigated by cell function experiments (Cell Counting Kit‑8, flow cytometry and Transwell assays). The targeting relationship between circRNA_0044556 and miR‑145 was investigated via bioinformatics analysis, dual‑luciferase reporter assay and RNA immunoprecipitation. The effects of the circRNA_0044556/miR‑145 axis on the TNBC cells were revealed by rescue experiments. Correlations among circRNA_0044556, miR‑145 and NRAS were analyzed by Pearson's correlation test. CircRNA_0044556 was highly expressed in TNBC tissues and cells with or without ADM‑resistance. The overexpression of circRNA_0044556 promoted cell viability, ADM‑resistance and migration, while inhibiting the apoptosis by sponging miR‑145. Upregulation of miR‑145 reversed the effects of circRNA_0044556 on the TNBC cells. CircRNA_0044556 was negatively correlated with miR‑145 yet positively correlated with NRAS, the target gene of miR‑145, in addition to the discovery suggesting the negative regulatory effects of circRNA_0044556 on miR‑145. CircRNA_0044556 diminished the sensitivity of TNBC cells to ADM via the miR‑145/NRAS axis.

Keywords: adriamycin; chemotherapy sensitivity; circular RNA_0044556; microRNA‑145; triple‑negative breast cancer.

Figure 1.

Expression of circRNA_0044556 in TNBC tissues and TNBC cells with or without ADM treatment. (A-D) The expression of circRNA_0044556 was quantified in normal mammary epithelial cells, TNBC tissues and TNBC cells with or without ADM-resistance by reverse transcription-quantitative PCR. GAPDH was used as the internal control. ***P<0.001 vs. Control; ^{^^^}P<0.001 vs. ADM-sensitive; ^###P<0.001 vs. MCF-10A; and ^&&&P<0.001 vs. MDA-MB-231. circRNA, circular RNA; TNBC, triple-negative breast cancer; ADM, adriamycin.

Figure 2.

CircRNA_0044556 plays a role in the viability of TNBC cells with or without ADM treatment. (A and B) Reverse transcription-quantitative PCR was performed to evaluate the expression of circRNA_0044556 following the overexpression or silencing of circRNA_0044556 in MDA-MB-231 cells with or without ADM treatment. GAPDH was used as the internal control. (C and D) A CCK-8 assay was used to determine the viability of parental MDA-MB-231 cells after gradient treatments of ADM, and the IC₅₀ value was calculated. (E and F) A CCK-8 assay was used to evaluate the viability of MDA-MB-231/ADM cells after gradient treatments of ADM, and the IC₅₀ value was calculated. ⁺P<0.05 and ⁺⁺⁺P<0.001 vs. NC; ^ΔΔP<0.01 and ^ΔΔΔP<0.001 vs. si-NC. circRNA, circular RNA; TNBC, triple-negative breast cancer; ADM, adriamycin; CCK-8, Cell Counting Kit-8; IC₅₀, half maximal inhibitory concentration; si-, small interfering; NC, negative control.

Figure 3.

CircRNA_0044556 plays a role in the apoptosis and migration of TNBC cells with or without ADM treatment. (A and B) Flow cytometry was employed to analyze the apoptosis following the overexpression or silencing of circRNA_0044556 in MDA-MB-231 cells with or without ADM treatment. (C and D) Transwell assay was performed to detect the migration capacity of transfected cells. ⁺⁺⁺P<0.001 vs. NC; and ^ΔΔΔP<0.001 vs. si-NC. circRNA, circular RNA; TNBC, triple-negative breast cancer; ADM, adriamycin; si-, small interfering; NC, negative control.

Figure 4.

MiR-145 is a novel target of circRNA_0044556 and its expression is identified in TNBC tissues and TNBC cells with or without ADM treatment. (A) The circInteractome website was used to predict the binding sites between circRNA_0044556 and miR-145. (B) Subsequently, a dual luciferase reporter assay was carried out for the validation that miR-145 was sponged by circRNA_0044556. (C) An RNA binding protein immunoprecipitation assay was performed to detect the association of miR-145 and circRNA_0044556 in TNBC cells. (D and E) The expression of miR-145 in TNBC tissues with or without ADM-resistance was determined by RT-qPCR. GAPDH was used as the internal control. (F) The correlation between circRNA_0044556 and miR-145 was analyzed with Pearson's correlation analysis. (G and H) The expression of miR-145 in normal mammary epithelial cells and TNBC cells with or without ADM-resistance was determined by RT-qPCR. GAPDH was used as the internal control. ^ΩΩΩP<0.001 vs. miR-NC; ***P<0.001 vs. Control; ^{^^^}P<0.001 vs. ADM-sensitive; ^###P<0.001 vs. MCF-10A and ^&&&P<0.001 vs. MDA-MB-231. miR, microRNA; circRNA, circular RNA; TNBC, triple-negative breast cancer; ADM, adriamycin; RT-qPCR, reverse transcription-quantitative PCR; NC, negative control.

Figure 5.

CircRNA_0044556 regulates the expression of miR-145 in TNBC cells with or without ADM treatment. (A and B) MDA-MB-231 cells were transfected with circRNA_0044556 overexpression plasmids, miR-145 mimic or both and MDA-MB-231/ADM cells were transfected with si-circRNA_0044556, miR-145 inhibitor or both, and the expression of miR-145 was calculated by reverse transcription-quantitative PCR. U6 was used as the internal control. ^‡‡‡P<0.001 vs. NC+MC; ^†††P<0.001 vs. NC+M; ^§§§P<0.001 vs. si-NC+IC; and ^@@@P<0.001 vs. si-NC+I. NC+MC: MDA-MB-231 cells were transfected with empty plasmids (negative control for circRNA_0044556) and mimic control (MC for miR-145); circRNA_0044556+MC: MDA-MB-231 cells were transfected with circRNA_0044556 overexpression plasmids and mimic control; NC+M: MDA-MB-231 cells were transfected with empty plasmids (negative control for circRNA_0044556) and miR-145 mimic; circRNA_0044556+M: MDA-MB-231 cells were transfected with circRNA_0044556 overexpression plasmids and miR-145 mimic; si-NC+IC: MDA-MB-231/ADM cells were transfected with si-NC (negative control for si-circRNA_0044556) and inhibitor control (IC for miR-145); si-circRNA_0044556+IC: MDA-MB-231/ADM cells were transfected with si-circRNA_0044556 and inhibitor control; si-NC+I: MDA-MB-231/ADM cells were transfected with si-NC and miR-145 inhibitor; si-circRNA_0044556+I: MDA-MB-231/ADM cells were transfected with si-circRNA_0044556 and miR-145 inhibitor. circRNA, circular RNA; miR, microRNA; TNBC, triple-negative breast cancer; ADM, adriamycin; si-, small interfering; NC, negative control; I, inhibitor; M, mimic; IC, inhibitor control; MC, mimic control.

Figure 6.

Regulatory role of the circRNA_0044556/miR-145 axis in the development of TNBC cells with or without ADM treatment. (A and B) Flow cytometry was employed to analyze the apoptosis after the overexpression or depletion of circRNA_0044556, miR-145 alone or in combination in MDA-MB-231 and MDA-MB-231/ADM cells. (C and D) The migratory capacities of TNBC cells after various transfections were evaluated by Transwell assay. ^‡‡‡P<0.001 vs. NC+MC; ^†††P<0.001 vs. NC+M; ^§P<0.05 and ^§§§P<0.001 vs. si-NC+IC; and ^@@@P<0.001 vs. si-NC+I. NC+MC: MDA-MB-231 cells were transfected with empty plasmids (negative control for circRNA_0044556) and mimic control (MC for miR-145); circRNA_0044556+MC: MDA-MB-231 cells were transfected with circRNA_0044556 overexpression plasmids and mimic control; NC+M: MDA-MB-231 cells were transfected with empty plasmids (negative control for circRNA_0044556) and miR-145 mimic; circRNA_0044556+M: MDA-MB-231 cells were transfected with circRNA_0044556 overexpression plasmids and miR-145 mimic; si-NC+IC: MDA-MB-231/ADM cells were transfected with si-NC (negative control for si-circRNA_0044556) and inhibitor control (IC for miR-145); si-circRNA_0044556+IC: MDA-MB-231/ADM cells were transfected with si-circRNA_0044556 and inhibitor control; si-NC+I: MDA-MB-231/ADM cells were transfected with si-NC and miR-145 inhibitor; si-circRNA_0044556+I: MDA-MB-231/ADM cells were transfected with si-circRNA_0044556 and miR-145 inhibitor. circRNA, circular RNA; miR, microRNA; TNBC, triple-negative breast cancer; ADM, adriamycin; si-, small interfering; NC, negative control; I, inhibitor; M, mimic; IC, inhibitor control; MC, mimic control.

Figure 7.

NRAS is targeted by miR-145 and the effect of circRNA_0044556/miR-145 axis on TNBC cells with or without ADM treatment is mediated by NRAS. (A) The potential binding site of NRAS for miR-145 was predicted by StarBase website. (B) A dual luciferase reporter assay was carried out for the validation that NRAS was targeted by miR-145. (C-F) The expression of NRAS was determined in TNBC tissues, ADM-sensitive TNBC tissues, ADM-resistant TNBC tissues, normal mammary epithelial cells and cells with or without ADM-resistance by reverse transcription-quantitative PCR. GAPDH was used as the internal control. (G and H) The correlation between miR-145 and NRAS, or between circRNA_0044556 and NRAS in TNBC was analyzed with Pearson's correlation analysis. (I and J) The expression of NRAS was detected after the overexpression or silencing of circRNA_0044556 in MDA-MB-231 and MDA-MB-231/ADM cells by reverse transcription-quantitative PCR. ^ΩΩΩP<0.001 vs. miR-NC; ***P<0.001 vs. Control; ^{^^^}P<0.001 vs. ADM-sensitive; ^###P<0.001 vs. MCF-10A; ^&&&P<0.001 vs. MDA-MB-231; ⁺⁺⁺P<0.001 vs. NC; and rrrP<0.001 vs. si-NC. NRAS, NRAS proto-oncogene, GTPase; miR, microRNA; circRNA, circular RNA; TNBC, triple-negative breast cancer; ADM, adriamycin.