circRNA_0025202 Regulates Tamoxifen Sensitivity and Tumor Progression via Regulating the miR-182-5p/FOXO3a Axis in Breast Cancer

Abstract

Tamoxifen is the most commonly used endocrine therapy for patients with hormone receptor (HR)-positive breast cancer. Despite its initial therapeutic efficacy, many patients eventually develop drug resistance, which remains a serious clinical challenge. To investigate roles of circular RNAs (circRNAs) in tamoxifen resistance, a tamoxifen-resistant MCF-7 cell line was established and screened for its circRNA expression profile by RNA sequencing. hsa_circ_0025202, a circRNA that was significantly downregulated, was selected for further investigation. Using a large cohort of clinical specimens, we found that hsa_circ_0025202 exhibited low expression in cancer tissues and was negatively correlated with lymphatic metastasis and histological grade. Gain- and loss-of-function assays indicated that hsa_circ_0025202 could inhibit cell proliferation, colony formation, and migration and increase cell apoptosis and sensitivity to tamoxifen. Bioinformatics and luciferase reporter assays verified that hsa_circ_0025202 could act as a miRNA sponge for miR-182-5p and further regulate the expression and activity of FOXO3a. Functional studies revealed that tumor inhibition and tamoxifen sensitization effects of hsa_circ_0025202 were achieved via the miR-182-5p/FOXO3a axis. Moreover, in vivo experiments confirmed that hsa_circ_0025202 could suppress tumor growth and enhance tamoxifen efficacy. Taken together, hsa_circ_0025202 served an anti-oncogenic role in HR-positive breast cancer, and it could be exploited as a novel marker for tamoxifen-resistant breast cancer.

Keywords: breast cancer; ceRNA; circRNA; hsa_circ_0025202; tamoxifen resistance.

Figure 1

The Characteristics of the hsa_circ_0025202 (A) Numbers of circRNA and backspliced reads identified in MCF7/TR and MCF7/P BC cell lines. (B) The proportions of circRNAs derived from different genomic regions. (C) Numbers of differently expressed circRNAs from each chromosome. (D) Top 10 differentially expressed circRNAs between MCF7/P and MCF7/TR cell lines. (E) qRT-PCR indicating the levels of hsa_circ_0025202 expression in MCF7/P and MCF7/TR cell lines. (F) Schematic illustrates the generation of hsa_circRNA_0025202. Blue arrows represent the detection of specific primers for hsa_circ_0025202 by qRT-PCR. (G) Gel electrophoresis shows that hsa_circ_0025202 can be amplified by divergent primers using total cDNA, but not genomic DNA (gDNA). Sanger sequencing validates its splicing junction. Actin served as a control. (H) qRT-PCR analysis of hsa_circ_0025202 and GAPDH expressions in MCF7 cells after treating with RNase R. (I) qRT-PCR data for the abundance of GAPDH mRNA and hsa_circRNA_0025202 in MCF7 cells at the indicated time points. (J) qRT-PCR analysis of hsa_circ_0025202, β-actin, and U6 in the cytoplasm and nucleus in MCF7 cells. (K) qRT-PCR shows differently expressed hsa_circRNA_0025202 in 41 normal and 230 tumor tissues. Data are presented as means ± SEM of three independent experiments. **p < 0.01.

Figure 2

Overexpression of hsa_circ_0025202 Reversed the Progressive Phenotype and TAM Resistance of MCF7/TR Cells In Vitro (A) qRT-PCR analysis of hsa_circ_0025202 and GAPDH expressions in MCF7/TR cells transfected with PLCDH and circ_0025202 ove. (B) MTT analysis of cell proliferation in MCF7/TR cells transfected with or without circ_0025202 ove at the indicated time points. (C) The colony formation, (D) cell migration, (E) cell apoptosis, and cell viability (F) and IC₅₀ (G) in MCF7/TR cells transfected with or without circ_0025202 ove. Data are presented as means ± SEM of three independent experiments. *p < 0.05; **p < 0.01; ***, ^###p < 0.001. Scale bar, 50 μm.

Figure 3

Knockdown of hsa_circ_0025202 Promotes Growth and Metastasis and Decreases TAM Sensitivity in HR-Positive BC Cells In Vitro (A) Schematic illustrates si-circ_0025202 targeted the backsplice junction of circ_0025202. (B) The expression of hsa_circ_0025202 and GAPDH in T47D and MCF7 cells was determined with qRT-PCR transfected with the negative control of siRNA (NC-si) and si-circ_0025202. (C–H) hsa_circ_0025202 was silenced in T47D and MCF7 cells. (C) MTT assay was conducted to evaluate cell proliferative ability. (D) Colony formation is shown in T47D and MCF7 cells. (E) Transwell migration was conducted to evaluate cell migration ability (magnification, 100× ). Scale bar, 50 μm. (F) MTT assay demonstrates cell viability of T47D and MCF7 cells treated together with TAM. (G) IC₅₀ assay shows the variation in TAM sensitivity. (H) Cells were treated with TAM (10 nM) or ethanol (control) and subjected to Annexin V-FITC and propidium iodine staining to detect apoptotic rate by flow cytometry. Data are presented as means ± SEM of at least three independent experiments. *, ^#p < 0.05; **p < 0.01; ***, ^###p < 0.001.

Figure 4

hsa_circ_0025202 Acts as an Efficient miRNA Sponge for miR-182-5p (A) A schematic illustration demonstrating the putative binding sites of the miRNAs associated with hsa_circ_0025202. (B) qRT-PCR data show expressions of the candidate miRNAs after hsa_circ_0025202 overexpression in MCF7 cells. (C) Expression of miR-182-5p in HR-positive BC tissues and adjacent normal tissues from TCGA data. (D) qRT-PCR for the expression of hsa_circ_0025202 and miR-182-5p in MCF7 cells after pulling down of Ago2 by RIP assay. (E) A schematic of wild-type (WT) and mutant (MUT) hsa_circ_0025202 luciferase reporter vectors. (F) The luciferase activity of WT hsa_circ_0025202 3′ UTR or mutant hsa_circ_0025202 3′ UTR after transfection with miR-182-5p mimics in 293T cells. Data are presented as means ± SEM of at least three independent experiments. *p < 0.05; **, ^##p < 0.01; ***, ^###p < 0.001.

Figure 5

hsa_circ_0025202 Sponges miR-182-5p to Regulate Tumor Progression and TAM Sensitivity of BC Cells (A) Cell proliferation, (B) migration, and (C) apoptosis of MCF7 cells after transfection with miR-182-5p mimics combined with or without circ_0025202 ove. (D) Cell proliferation, (E) migration, and (F) apoptosis of MCF7/TR cells after transfection with miR-182-5p inhibitor combined with or without si-circ_0025202. MTT data for the cell viability (G) and IC₅₀ (H) of MCF7 cells treated with TAM after transfection with miR-182-5p mimics combined with or without circ_0025202 ove. MTT data for the cell viability (I) and IC₅₀ (J) of MCF7/TR cells treated with TAM after transfection with miR-182-5p inhibitor combined with or without si-circ_0025202. Data are presented as means ± SEM of at least three independent experiments. *, ^#p < 0.05; **, ^##p < 0.01; ***, ^###p < 0.001. Scale bar, 50 μm.

Figure 6

FOXO3a Is a Direct Target Gene of miR-182-5p and Is Regulated by hsa_circ_0025202 (A) Venn diagram represents the mutual candidate target genes of miR-182-5p identified by miRWalk, miRDB, TargetScan, and miRanda. (B and C) FOXO3a expression assessed by qRT-PCR (B) and western blot (C), respectively, in MCF7 and T47D cells after transfection with miR-182-5p mimics and circ_0025202 ove. (C) The quantification of the bands is shown. (D) Potential binding sites of miR-182-5p with FOXO3a 3′ UTR. (E) The luciferase activity of WT FOXO3a 3′ UTR or mutant FOXO3a 3′ UTR of two potential binding sites after transfection with miR-182-5p mimics in HEK293T cell lines. (F) MTT data for the proliferation of MCF7 and T47D cell lines treated with TAM after transfection of si-FOXO3a. (G) MTT data for the cell viability and IC₅₀ of MCF7 and T47D cell lines treated with TAM after transfection of si-FOXO3a. (H) Annexin V-FITC assay was used to assess cell apoptosis in MCF7 and T47D cells after transfection of si-FOXO3a. Data are presented as means ± SEM of at least three independent experiments. *, ^#p < 0.05; **, ^##p < 0.01; ***, ^###p < 0.001.

Figure 7

Overexpression of hsa_circ_0025202 Suppressed the Growth and TAM Resistance of BC Xenografts (A) Images of the xenograft tumors obtain from BALB/c nude mice at the endpoint. (B) Growth curve of tumor volume. The volumes were measured every 5 days after the initial 2 weeks. (C) Weights of xenograft tumors at the endpoint. (D) FOXO3a, Ki67, and cleaved caspase-3 staining by IHC of the xenograft tumors. Scale bar, 100 μm. Data are presented as means ± SEM of at least three independent experiments. **p < 0.01; ***, ^###p < 0.001.