Exosomal MicroRNA-221-3p Confers Adriamycin Resistance in Breast Cancer Cells by Targeting PIK3R1

Abstract

Drug resistance in breast cancer (BC) cells continues to be a stern obstacle hindering BC treatment. Adriamycin (ADR) is a frequently employed chemotherapy agent used to treat BC. The exosomal transfer of microRNAs (miRNAs) has been reported to enhance the drug-resistance of BC cells. Herein, we first sought to elucidate the possible role of the exosomal transfer of miR-221-3p in the drug resistance of MCF-7 cells to ADR. Differentially expressed genes (DEGs) were initially screened through microarray analysis in BC drug resistance-related datasets. Next, the expression of miR-221-3p and phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) was quantified in ADR-resistant MCF-7 (MCF-7/ADR) and ADR-sensitive MCF-7 (MCF-7/S) cell lines, after which exosomes were separated and identified in each cell line. Target relationship between miR-221-3p and PIK3R1 was validated by a dual-luciferase reporter assay. Next, the expression of miR-221-3p and PIK3R1 was altered to clarify their effects on the resistance of MCF-7 cells to ADR in vitro and in vivo. PIK3R1 was identified as a BC drug resistance-related DEG, with the regulatory miR-221-3p subsequently obtained. Moreover, the MCF-7/ADR cells exhibited a low expression of PIK3R1 and a high expression of miR-221-3p. Notably, PIK3R1 was identified as a target gene of miR-221-3p. The overexpression of miR-221-3p in MCF-7/ADR cell-derived exosomes promoted ADR resistance in MCF-7/S cells via the PI3K/AKT signaling pathway. The in vitro results were reproducible in in vivo assays. Taken together, drug-resistant BC cell-derived exosomal miR-221-3p can promote the resistance of BC cells to ADR by targeting PIK3R1 via the PI3K/AKT signaling pathway in vitro and in vivo. These findings provide encouraging insights and provide perspectives for further investigation into the BC drug resistance mechanism.

Keywords: adriamycin; breast cancer; drug resistance; exosomes; microRNA-221-3p; phosphoinositide-3-kinase regulatory subunit 1.

Figure 1

PIK3R1 is poorly expressed in drug-resistant BC cells. (A) A volcano map depicting the expression of DEGs associated to the BC drug resistance in the GSE76540 dataset. X-axis indicated log10 p-value, and Y-axis represented logFC. Each point indicated a gene (red points represented upregulated DEGs in drug-resistant samples and green points represented downregulated DEGs in drug-resistant samples). (B) PPI network of DEGs. Each circle indicated a gene, and the line between circles represented their interaction. The darker color indicated a higher core degree. (C) Sub-network of PIK3R1 interacting with PPI network. (D) Expression of UBB, GNGT1, PIK3R1, GNB2, and ESR1 in normal MCF-10A and ADR-sensitive MCF-7/S cells detected by RT-qPCR. (E) PIK3R1 expression in MCF-10A, MCF-7/S, and ADR-resistant MCF-7/ADR cells measured by RT-qPCR. (F) western blot analysis of PIK3R1 protein in MCF-10A, MCF-7/S, and MCF-7/ADR cells. Measurement data were expressed as mean ± standard deviation. Data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, compared with MCF-10A cells; #p < 0.05, compared with MCF-7/S cells. Each experiment was repeated three times independently.

Figure 2

PIK3R1 overexpression reduces cell viability and drug resistance but increases cell apoptosis. (A) PIK3R1 mRNA expression in MCF-7/S cells detected by RT-qPCR. (B) Western blot analysis of PIK3R1 protein in MCF-7/S cells. (C) PIK3R1 mRNA expression in MCF-7/ADR cells determined by RT-qPCR. (D) Western blot analysis of PIK3R1 protein in MCF-7/ADR cells. (E) IC₅₀-value in MCF-7/S cells with ADR measured by MTT. (F) IC₅₀-value in MCF-7/ADR cells with ADR calculated by MTT. (G) viability of MCF-7/S cells analyzed by MTT. (H) Viability of MCF-7/ADR cells analyzed by MTT. (I) Apoptosis of MCF-7/S cells examined using flow cytometry. (J) apoptosis of MCF-7/ADR cells examined using flow cytometry. Measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test while data comparison at different time points was conducted by repeated measures ANOVA, followed by Bonferroni post-hoc test. *p < 0.05, compared with the oe-NC-transfected cells; #p < 0.05, compared with sh-NC-transfected cells. Each experiment was repeated three times independently.

Figure 3

MCF-7/S cells endocytose MCF-7/ADR cell-derived exosomes. (A) Exosome identified by a TEM (200 nm). (B) Diameter of exosomes measured by dynamic light scattering. (C) Western blot analysis of exosomal surface markers CD63 and HSP70 proteins. Lane 1: isolated exosome; lane 2: supernatant of isolated exosome. (D) CD63 content determined by flow cytometry. (E) Glioma cell endocytosing exosome analyzed under an inverted fluorescence microscope (200×). The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test. Each experiment was repeated three times independently.

Figure 4

MCF-7/ADR cell-derived exosomes transfer drug resistance to MCF-7/S cells. (A) IC₅₀-value in MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes detected by MTT assay. (B) Viability in MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes detected by MTT assay. (C) Apoptosis of MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes determined by flow cytometry. (D) PIK3R1 expression in MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes measured by RT-qPCR. (E) Western blot analysis of PIK3R1 protein in MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes. The measurement data were expressed as mean ± standard deviation. Data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test while data comparison at different time points was analyzed by repeated measures ANOVA, followed by Bonferroni post-hoc test. *p < 0.05, compared with MCF-7/S cells; #p < 0.05, compared with MCF-7/S cells co-cultured with MCF-7/ADR cell-derived exosomes. Each experiment was repeated three times independently.

Figure 5

BC cell exosomal miR-221-3p targets PIK3R1. (A) Upstream regulatory miRNAs of PIK3R1 predicted using the mirDIP and TargetScan databases. Three circles were indicative of the results obtained from the EVmiRNA, mirDIP, and TargetScan databases, respectively, and the middle part denoted the intersected results of these three databases. (B) miRNA expression in MCF-10A, MCF-7/S, and MCF-7/ADR cells detected by RT-qPCR. *p < 0.05 against MCF-10A cells. (C) Binding sites between PIK3R1 and miR-221-3p analyzed using an online software. (D) miR-221-3p expression in exosomes derived from ADR-sensitive and ADR-resistant cells analyzed by RT-qPCR. (E) The binding of PIK3R1 to miR-221-3p verified by dual-luciferase reporter assay. (F) PIK3R1 expression in MCF-7/ADR cells transfected with miR-221-3p mimic or inhibitor measured by RT-qPCR. (G) Western blot analysis of PIK3R1 protein in MCF-7/ADR cells transfected with miR-221-3p mimic or inhibitor. The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test, while data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test. *p < 0.05, compared with mimic NC-transfected cells; #p < 0.05, compared with inhibitor NC-transfected cells. Each experiment was repeated three times independently.

Figure 6

Exosomal miR-221-3p promotes BC cell drug resistance by targeting PIK3R1 and inhibiting the PI3K/AKT signaling pathway. (A) Expression of miR-221-3p and PIK3R1 in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor detected by RT-qPCR. (B) Western blot analysis of PIK3R1 protein in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor. (C) IC₅₀-value in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (D) Viability of MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (E) Apoptosis of MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor measured by MTT assay. (F) Western blot analysis of PI3K, and AKT as well as the extent of AKT and PI3K phosphorylation in MCF-7/S cells treated with MCF-7/ADR cell-derived exosomes co-cultured with miR-221-3p mimic or inhibitor. The measurement data were expressed as mean ± standard deviation. Data comparison between two groups was conducted by unpaired t-test, while data comparison at different time points was conducted by repeated measures ANOVA, followed by Bonferroni post-hoc test. *p < 0.05, compared with the cells treated with MCF-7/ADR cell-derived exosomes co-cultured with mimic-NC; #p < 0.05, compared with the cells treated with MCF-7/ADR cell-derived exosomes co-cultured with inhibitor-NC. Each experiment was repeated three times independently.

Figure 7

Inhibition of exosomal miR-221-3p enhances the suppressive role of ADR in tumor formation in nude mice. (A) Representative images of tumors formed in nude mice inoculated with MCF-7/ADR cell-derived exosomes treated with ADR or both miR-221-3p inhibitor and ADR. (B) A tumor growth curve of xenograft tumor of mice inoculated with MCF-7/ADR cell-derived exosomes treated with ADR or both miR-221-3p inhibitor and ADR. (C) Quantitation of tumor volume in nude mice inoculated with MCF-7/ADR cell-derived exosomes treated with ADR or both miR-221-3p inhibitor and ADR. (D) Quantitation of tumor weight in nude mice inoculated with MCF-7/ADR cell-derived exosomes treated with ADR or both miR-221-3p inhibitor and ADR. (E) Cell apoptosis in tumor of mice inoculated with MCF-7/ADR cell-derived exosomes treated with ADR or both miR-221-3p inhibitor and ADR detected by flow cytometry. (F) Western blot analysis of PI3K, and AKT as well as the extent of AKT and PI3K phosphorylation in tumor tissues of nude mice. The measurement data were described as mean ± standard deviation. Data comparison among multiple groups was conducted by one-way ANOVA with Dunnett's post-hoc test, while data comparison at different time points was conducted by repeated measures ANOVA, followed by Bonferroni post-hoc test. N = 6. *p < 0.05, compared with the mice following PBS treatment; #p < 0.05, compared with ADR treatment. &p < 0.05, compared with the mice inoculated with MCF-7/ADR cell-derived exosomes treated with inhibitor-NC. Each experiment was repeated three times independently.

Figure 8

The graph of the molecular mechanism of miR-221-3p in ADR-resistant BC cells. In ADR-resistant BC cells, miR-221-3p promotes drug resistance by negatively regulating PIK3R1, thereby promoting BC cell viability and inhibiting apoptosis. In addition, miR-221-3p can be packaged into the exosomes derived from ADR-resistant BC cells, and transferred to ADR-sensitive BC cells.