Epithelial membrane protein 3 functions as an oncogene and is regulated by microRNA-765 in primary breast carcinoma

Abstract

Epithelial membrane protein 3 (EMP3) is a transmembrane signaling molecule, which is important in the regulation of apoptosis, differentiation and invasion of cancer cells. However, the specific function and regulatory mechanism of EMP3 in primary breast carcinoma remain to be elucidated. In the present study, the mRNA and protein levels of EMP3 were observed to be upregulated in primary breast carcinoma tissues, compared with normal tissues. It was hypothesized that the overexpression of EMP3 was correlated with the downregulation of microRNA‑765 (miR‑765), an underexpressed miRNA in primary breast carcinoma tissues. Functional analysis demonstrated that EMP3 was regulated by miR‑765 through binding to its 3'untranslated region. In addition, the knockdown of EMP3 and miR‑765 had similar effects on the inhibition of proliferation and invasion in SK‑BR‑3 cells. These results provided novel insight into the regulatory mechanism of EMP3 in primary breast carcinoma.

Figure 1

Differential expression of EMP3 in primary breast carcinoma tissues, compared with adjacent normal tissues. (A) Relative mRNA expression levels of EMP3 in six breast cancer tissues and paired adjacent normal tissues. The mRNA levels were detected using reverse-transcription-quantitative polymerase chain reaction. Combination analysis represents the mean EMP3 level in the six samples. (B) Protein expression levels of EMP3 in primary breast tissue and normal tissues using western blot analysis. All data are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, vs. control. EMP3, epithelial membrane protein 3; T, tumor; N, normal.

Figure 2

miR-765 directly targets EMP3 and represses mRNA levels of EMP3 in SK-BR-3 cells. (A) Schematic representation of the miRNA target prediction algorithm (miRwalk) screening of the potential miRNAs targeting the EMP3 mRNA 3′UTR. (B) Luciferase constructs were co-transfected with the luciferase construct (pGL3/luciferase EMP3-3′UTR) and five miR mimics, including a control mimic. Luciferase activity was determined 48 h after transfection, and normalized to the control. (C) Luciferase constructs were transfected into cells transduced with the miR-765 mimics and control. Luciferase activity was determined 48 h after transfection. The ratio of normalized to control luciferase activity is presented. (D) miR-765 and miR-765 inhibitor were transfected into SK-BR-3 cells and the mRNA level of EMP3 was measured. Actin was used as a loading control. The level of EMP3 in the control and inhibitor control group were set as 1.0. All the data are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, vs. control. miR-765, microRNA-765; EMP3, epithelial membrane protein 3; UTR, untranslated region; WT, wild-type; Mut, mutant.

Figure 3

Determination of the expression of miR-765 in primary breast tissues and cell lines. (A) Relative expression levels of miR-765 in primary breast carcinoma tissues, compared with corresponding non-tumor lung tissues. The expression of miR-765 was normalized to U6. (B) Relative expression levels of miR-765 was inversely associated with EMP3 in four breast cancer cell lines (MDA-MB-231, MCF-7, MDA-MB-453 and SK-BR-3). miRNA/miR, microRNA; EMP3, epithelial membrane protein 3.

Figure 4

EMP3 modulation accounts for the anti-proliferative effects of miR-765 in SK-BR-3 cells. (A) Western blotting for the protein levels of EMP3 in SK-BR-3 cells following 48 h of miR-765 and EMP3 shRNA transfection. (B) Effects of miR-765 and shRNA EMP3 on the proliferation of SK-BR-3 cells. (C) Effect of miR-765 and shRNA EMP3 on the cell invasive ability of SK-BR-3 cells. The arrows indicate invaded cells. Scale bar=20 µm. (D) Effect of miR-765 and shRNA EMP3 on the migratory ability of SK-BR-3 cells. All data are expressed as the mean ± standard deviation of three independent experiments. ^*P<0.05, vs. control. EMP3, epithelial membrane protein 3; miR, microRNA; shRNA, short hairpin RNA.

Figure 5

Schematic representation of the hypothetical molecular mechanism of EMP3 regulation in primary breast carcinoma. Top line: DNA methylation regulation mechanism had no effect on EMP3 upregulation in primary breast carcinoma. Bottom line: miR-765 was involved in EMP3 upregulation in primary breast carcinoma. EMP3, epithelial membrane protein 3; miR, microRNA; CDS, coding sequence.