Co-treatment with miR-21-5p inhibitor and Aurora kinase inhibitor reversine suppresses breast cancer progression by targeting sprouty RTK signaling antagonist 2

Abstract

Numerous studies have reported the regulatory effects of miR-21-5p and reversine in human breast cancer (HBC). However, the mechanism of reversine and miR-21-5p has not been fully investigated in HBC. The aim of the current study was to assess the mechanism of action of reversine, with or without miR-21-5p, in HBC progression. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and Western blot results confirmed the upregulation of miR-21-5p and downregulation of sprouty RTK signaling antagonist 2 (SPRY2) in HBC. Bioinformatics analysis and luciferase assay identified the correlation between miR-21-5p and SPRY2. Cell function experiment results indicated a decrease in migration, proliferation, and invasion of HBC cells treated with miR-21-5p inhibitor and reversine; however, an increase in apoptosis was observed in these cells. Apoptotic ability was more enhanced and migration, proliferation, and invasion were more impaired in HBC cells treated with both miR-21-5p inhibitor and reversine than in those treated individually with either inhibitors. SPRY2, downstream of miR-21-5p, participated in HBC progression with reversine. Overall, our study proved that combining the miR-21-5p inhibitor with reversine produced a synergistic effect by regulating SPRY2, thereby limiting HBC progression. This knowledge might offer insights into the clinical therapy of HBC.

Keywords: Human breast cancer; SPRY2; miR-21-5p; reversine.

Figure 1.

Reversine suppressed breast cancer cells proliferation, migration and invasion while exacerbated their apoptosis in a dose-dependent manner. (a) The human breast cancer cell lines MDA-MB-231 and MCF-7 were treated with various dosages of reversine for 24 h and 48 h, and the CCK-8 assay was used to evaluate cell viability. (b) The cell viability of MDA-MB-231 and MCF-7 cell lines under the treatment of 0 μM, 0.15 μM and 0.3 μM reversine for 24 h, 48 h and 72 h was determined by CCK-8 assay. (c) BrdU assay indicated the cell proliferation of MDA-MB-231 and MCF-7 treated with 0 μM, 0.15 μM and 0.3 μM reversine for 48 h. (d) Apoptosis rate was examined in MDA-MB-231 and MCF-7 treated with 0 μM, 0.15 μM and 0.3 μM reversine for 48 h. (e) Wound healing assay showed cell migration ability of MDA-MB-231 and MCF-7 treated with 0 μM, 0.15 μM and 0.3 μM reversine for 48 h. (f) Transwell assay indicated cell invasion capacity of MDA-MB-231 and MCF-7 treated with 0 μM, 0.15 μM and 0.3 μM reversine for 48 h. * P < 0.05, ** P < 0.001 compared to 0 μM group.

Figure 2.

MiR-21-5p was significantly upregulated in breast cancer cells and tissues. (a) Relative miR-21-5p expression was detected by RT-qPCR in breast cancer tissues and adjacent normal tissues. (b) Relative miR-21-5p expression was detected by RT-qPCR in breast cancer cell lines (MDA-MB-231, SKBr-3, MCF-7 and BT474) and normal breast cell line (MCF-10A). (c) RT-qPCR analysis of miR-21-5p expression in MDA-MB-231 and MCF-7 cell lines treated with 0 μM, 0.15 μM and 0.3 μM reversine for 48 h. (d) RT-qPCR analysis of miR-21-5p expression in MDA-MB-231 and MCF-7 cell lines treated with negative control (NC), miR-21-5p inhibitor (miR-inhibitor) or miR-21-5p inhibitor together with 0.3 μM reversine for 48 h. Blank indicated untreated group. * P < 0.05, ** P < 0.001 compared to MCF-10A or 0 μM group or Blank group.

Figure 3.

Reversine and miR-21-5p inhibitor co-treatment showed synergistically restrictive effect on the cell proliferation, migration, invasion and enhanced effect on the apoptosis of breast cancer cell. For all the experiments, MDA-MB-231 and MCF-7 cell line were treated with negative control (miR-NC), miR-21-5p inhibitor alone (miR-inhibitor), 0.3 μM reversine alone (0.3 μM) or miR-21-5p inhibitor together with 0.3 μM reversine (0.3 μM + inhibitor group) for 48 h, respectively. Blank indicated untreated group. (a) The cell viability of MDA-MB-231 and MCF-7 cell lines under various treatment were determined by CCK-8 assay. (b) BrdU assay indicated the cell proliferation of MDA-MB-231 and MCF-7 cells under various treatment. (c) Apoptosis rate was examined in MDA-MB-231 and MCF-7 cells under various treatment. (d) Wound healing assay was performed to observe cell migration ability of MDA-MB-231 and MCF-7 cells under various treatment. (e) Transwell assay indicated cell invasion capacity of MDA-MB-231 and MCF-7 cells under various treatment. * P < 0.05, ** P < 0.001 compared to Blank group.

Figure 4.

Reversine combined with miR-21-5p inhibitor weakens the tumor growth of HBC in vivo (a) Tumor growth curves in xenograft formation assay, and the representative images of xenograft tumors dissected from the nude mice. (b) The tumor weight of xenograft tumors dissected from the nude mice. ** P < 0.001 compared to antagomiR-NC group. ## P < 0.001 compared to 1.0 mg/kg+antagomiR group.

Figure 5.

The anti-tumor effect of co-treatment of reversine and miR-21-5p inhibitor on HBC cells by targeting SPRY2. (a) 105 common genes were overlapped from starBase and GSE124646. starBase, a tool for predicting the targets of miR-21-5p. GSE124646, a mRNA microarray for screening the downregulated genes in HBC samples. (b) Seven genes were identified to be related to cell proliferation and cell migration by STRING analysis. (c) The negative correlation between SPRY2 and miR-21-5p in BRCA samples by starBase analysis. BRCA, breast invasive carcinoma. (d) starBase predicted the binding sites between SPRY2 3ʹUTR and miR-21-5p. (e) Luciferase assay identified the target relationship between SPRY2 3ʹUTR and miR-21-5p. (f) SPRY2 expression reduced in tumor samples compared with adjacent normal samples. (g) The negative correlation between SPRY2 and miR-21-5p in tumor samples by Pearson’s correlation analysis. (h) The upregulation of SPRY2 in HBC cells treated with reversine. ** P < 0.001 compared to 0 μM group. (i) The upregulation of SPRY2 in HBC cells treated with reversine and miR-21-5p inhibitor. ** P < 0.001 compared to Blank group.