MiR-363-3p induces tamoxifen resistance in breast cancer cells through PTEN modulation

Abstract

Nowadays, the investigation for overcoming tamoxifen (TAM) resistance is confronting a considerable challenge. Therefore, immediate attention is required to elucidate the mechanism underlying TAM resistance in breast cancer. This research primarily aimed to define how miRNA-363-3p facilitates resistance to TAM in breast cancer. High-throughput miRNA sequencing was performed using RNAs prepared from breast cancer MCF-7 cells and TAM-resistant MCF-7 cells (MCF-7-TAM). An increase in miRNA-363-3p levels was observed in MCF-7-TAM cells. In MCF-7 cells, miRNA-363-3p directly targeted and negatively regulated phosphatase and tensin homolog (PTEN). Reduction of miRNA-363-3p retarded cell growth and accelerated cell apoptosis, thereby enhancing the sensitivity of TAM. Moreover, analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway showed significant enrichment of target genes within the phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. Ultimately, miR-363-3p decreased the responsiveness of breast cancer cells to TAM by targeting and suppressing PTEN through a mechanism associated with the PI3K-Akt pathway. Therefore, these results suggest that miR-363-3p-dependent PTEN expression contributes to the mechanisms underlying breast cancer endocrine resistance.

Keywords: Breast cancer; Hsa-miR-363-3p; PI3K/AKT signaling pathway; PTEN; TAM resistance.

Fig. 1

Differential miRNA expression between MCF-7 as well as MCF-7-TAM cells. (A) Colony formation assay and CCK-8 assay. (B) Comparison rate analysis chart. Match rate (%): the percentage of clean reads that matched with the genomic reference sequence (one mismatch allowed); Non-match rate (%): the percentage of clean reads that did not match with the genomic reference sequence (greater than one mismatch). (C) Volcano plot of variance analysis results. The horizontal coordinates represent ploidy changes in miRNA expression in different experimental groups/samples. The vertical axis represent the statistical significance of changes in miRNA expression. The scattered dots represent individual miRNAs, the gray dots indicate miRNAs that are not significantly differentially expressed, the yellow dots indicate significantly upregulated miRNAs and the blue dots indicate significantly downregulated miRNAs. (D) Differential miRNA clustering analysis. Columns indicate different samples, rows indicate different genes, yellow represents upregulated genes and blue represents downregulated genes. (E) The overexpression of miR-363-3p in MCF-7-TAM cells was verified by RT-qPCR. ^*P < 0.05,^**P < 0.01; ^***P < 0.001. TAM, tamoxifen; RT-qPCR, real-time fluorescence quantitative polymerase chain reaction.

Fig. 2

GO and KEGG enrichment analysis. (A) Bar graph depiction of the GO enrichment analysis of the target genes of differentially expressed miRNAs. GO, Gene Ontology. The different colors signify the three broad categories of GO enrichment: biological processes, cellular components and molecular function. (B) KEGG enrichment analysis. KEGG, Kyoto Encyclopedia of Genes and Genomes. The size of each dot indicates the number of genes enriched in that GO subcategory. Larger dots indicate more genes enriched in that GO subcategory and vice versa.

Fig. 3

Alterations in cell proliferation after downregulating miR-363-3p. (A) Validation of successful transfection targeting miR-363-3p via RT-qPCR. (B) Alterations in cell proliferation subsequent to the knockdown of miR-363-3p and treatment with TAM. (C) Alterations in the number of colonies formed of treatment with si-miR-363-3p as well as TAM. ^**P < 0.01; ^***P < 0.001. Scale bar: 3 mm.

Fig. 4

Impacts of the knockdown of miR-363-3p on cell proliferation and apoptosis. The alterations in the cell proliferation marker Ki-67 (A) and the apoptosis marker caspase-3 (B) subsequent to the knockdown of miR-363-3p and the treatment of TAM were examined via immunofluorescence (IF). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. Scale bar: 50 µM.

Fig. 5

Alterations in cell proliferation after miR-363-3p upregulation. (A) Verification of successful transfection for overexpressing miR-363-3p. (B) Alterations in cell proliferation. (C) Changes in the colony formation ability. ^**P < 0.01; ^***P < 0.001. Scale bar: 3 mm.

Fig. 6

Impacts of the overexpression of miR-363-3p on cell proliferation and apoptosis. The alterations in the cell proliferation marker Ki-67 (A) and the apoptosis marker caspase-3 (B) subsequent to the overexpression of miR-363-3p and the treatment of TAM were examined via IF. ^**P < 0.01, ^***P < 0.001. Scale bar: 50 μm.

Fig. 7

miR-363-3p promoted PI3K–AKT signaling by inhibiting PTEN in breast cancer cells. (A) Dual luciferase assay verified that PTEN was a gene target of miR-363-3p. (B) PTEN protein expression in breast cancer cells was detected by Western blotting. (C) The expression of proteins related to the PI3K–AKT pathway was detected in breast cancer cells by Western blotting. ^***P < 0.001. PI3K, phosphoinositide 3-kinase.