MiR-654-5p attenuates breast cancer progression by targeting EPSTI1

Abstract

MicroRNAs (miRNAs) dysregulation is a common event in a variety of human diseases including breast cancer. However, clinical relevance and biological role of miR-654-5p in the progression of breast cancer remain greatly elusive. Herein, the expression levels of miR-654-5p were aberrantly downregulated in human breast cancer specimens and four breast cancer cell lines. Low expression of miR-654-5p was strongly associated with advanced TNM stage and lymph node metastasis as well as a poor survival. Functional analysis showed that miR-654-5p overexpression inhibited cell growth and invasion, and induced cell apoptosis in two aggressive breast cancer cells. Further studies demonstrated that Epithelial stromal interaction 1 (EPSTI1) was a direct target gene of miR-654-5p and showed an inverse correlation with miR-654-5p expression. Forced expression of EPSTI1 could abrogate the inhibitory effect of miR-654-5p on the growth and invasion of breast cancer cells as well as apoptosis-induced ability. In conclusion, the present study highlights that miR-654-5p acts as a tumor suppressor in breast cancer through directly targeting EPSTI1, and their functional regulation may open a novel avenue with regard to the therapeutic target for breast cancer.

Keywords: EPSTI1; breast cancer; miR-654-5p; miRNA; survival.

Figure 1

Relative expression of miR-654-5p in breast cancer tissues and cell lines as well as its correlation with overall survival of breast cancer patients. (A) MiR-654-5p expression was measured by qPCR and normalized to U6 expression in 110 paired breast cancer specimens, *P<0.001. (B) qPCR of miR-654-5p expression in HBL-100, MCF-7, MDA-MB-231, MDA-MB-468, and BT-549 cells and U6 acted as reference, *P<0.01. (C) The optimal cutoff value of miR-654-5p was determined by ROC analysis according to overall survival. Kaplan-Meier survival curve and log-rank test were employed to evaluate the associations between miR-654-5p expression (D), TNM stage (E), lymph node metastasis (F), and overall survival in breast cancer patients.

Figure 2

Ectopic expression of miR-654-5p inhibits cell proliferation, invasion and induces cell apoptosis of breast cancer cells. Cell proliferation was performed in MDA-MB-468 (A) and BT-549 (B) cells stably transfected with expression vectors carrying miR-654-5p or mi-NC. (C) Cell apoptosis was examined by flow cytometry with Annexin V-FITC and propidium iodide staining. (D) Cell apoptotic rate was counted according to summation of the second quadrant and fourth quadrant. (E) Matrigel invasion assays were conducted to determine breast cancer cell invasive ability. (F) The number of invaded cells was calculated, mean ± SD, *P<0.01.

Figure 3

MiR-654-5p targets EPSTI1 in breast cancer cell lines. A. Putative miR-654-5p binding sites in the 3’-UTR of EPSTI1 mRNA. B. The wild type and mutant of 3’-UTR reporter vectors and control group were co-transfected into breast cancer cells with miR-654-5p or miR-NC. The relative luciferase activities were evaluated. C. Cellular lysates from breast cancer cells were used to measure EPSTI1 expression by western blot. D. The expression of EPSTI1 mRNA was examined by qPCR in the transfected breast cancer cells. E. The correlation analysis of miR-654-5p and EPSTI1 mRNA expression levels in 70 breast cancer specimens. All experiments were performed in triplicate, mean ± SD, *P<0.01.

Figure 4

EPSTI1 attenuates the suppressive effect of miR-654-5p on cell proliferation and invasion as well as apoptosis-induced ability. A. Western blot analysis of EPSTI1 protein expression in breast cancer cells carrying miR-NC or miR-654-5p transfected with either pcDNA3.1 or pcDNA3.1-EPSTI1, GAPDH was used as a loading control. B. CCK-8 assay detecting the proliferation of breast cancer cells carrying miR-NC or miR-654-5p transfected with either pcDNA3.1 or pcDNA3.1-EPSTI1. C. Cell apoptosis was examined in breast cancer cells carrying miR-NC or miR-654-5p transfected with either pcDNA3.1 or pcDNA3.1-EPSTI1 by flow cytometry. D. Matrigel invasion assay evaluating invasion of breast cancer cells carrying miR-NC or miR-654-5p transfected with either pcDNA3.1 or pcDNA3.1-EPSTI1. Each assay was repeated three times. *P<0.05.