Silencing the COPB2 Gene Decreases the Proliferation, Migration and Invasion of Human Triple‑Negative Breast Cancer Cells
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EXPERIMENTAL AND THERAPEUTIC MEDICINE 22: 792, 2021 Silencing the COPB2 gene decreases the proliferation, migration and invasion of human triple‑negative breast cancer cells WENCHENG WU1*, CHENYU WANG1*, FENGXIA WANG1, YAN WANG2, YANLING JIN1, JING LUO1, MIN WANG1, CHENLI ZHANG1, SHUYA WANG1, FANGFANG ZHANG1 and MIN LI1,3 1Department of Pathology, School of Basic Medical Sciences, Lanzhou University; 2Gansu Provincial Hospital; 3Gansu Provincial Key Laboratory of Preclinical Study for New Drug Development, Lanzhou University, Lanzhou, Gansu 730000, P.R. China Received November 11, 2020; Accepted April 23, 2021 DOI: 10.3892/etm.2021.10224 Abstract. Triple‑negative breast cancer (TNBC) is highly develops in young women and is phenotypically defined as invasive, has a high rate of recurrence and is associated with a the lack of receptors for estrogen (ER), progesterone (PR) poor clinical outcome when compared with non‑TNBC due to a and human epidermal growth factor receptor 2 (HER‑2) (3). lack of effective and targeted treatments. The coatomer protein Cells from TNBC are more invasive, prone to lymph node complex subunit β2 (COPB2) is upregulated in various types of metastasis and are associated with poor clinical outcomes, malignant cancer. The present study demonstrated that COPB2 when compared with cells from non‑TNBC (4‑6). TNBC cells expression levels were significantly upregulated in breast respond poorly to endocrine and anti‑HER‑2 therapeutic strate‑ carcinoma HS‑578T cells (clonal cells originating from TNBC) gies, as they lack the receptors targeted by these therapies and, when compared with non‑TNBC MCF‑7 cells. HS‑578T cells thus, surgical treatment is preferred, followed by postoperative also exhibited higher rates of proliferation, invasion and chemotherapy or radiotherapy (2). Ongoing clinical studies transendothelial migration when compared with MCF‑7 cells. are evaluating the efficacy of targeting PI3K and inhibiting Moreover, it was identified that genetically silencing theCOPB2 EGFR (7), but their efficacies remain unknown. gene using a lentivirus‑short hairpin RNA inhibited the prolif‑ BC cells invade and migrate to surrounding or distant erative, colony formation, migratory and invasive properties of tissues via the process of epithelial‑mesenchymal transi‑ the TNBC HS‑578T cells. Mediation of the COPB2 silencing tion (EMT) (8), which converts cancerous epithelial cells effect may be associated with regulating the phosphorylation to mesenchymal cells under defined physiologic or patho‑ of serine/threonine kinase AKT in the PI3K/AKT signaling logical conditions (9). EMT is characterized by the decreased pathway. These results suggested the importance of COPB2 expression of epithelial markers, such as E‑cadherin (E‑Ca) in promoting the proliferation of TNBC cells and identified and increased expression of mesenchymal markers, such as COPB2 as a potential novel therapeutic target. N‑cadherin (N‑Ca) and vimentin (10,11). While being homolo‑ gous and belonging to the same cadherin superfamily, E‑Ca Introduction is primarily expressed on epithelial cells and is reported to inhibit cancer cell invasion (12,13), while N‑Ca is one of the Breast cancer (BC) is a common malignancy in females, major cadherins expressed on mesenchymal cells. Therefore, with an estimated 2.1 million new cases (11.6%) and 626,679 switching expression from E‑Ca to N‑Ca is a key cellular deaths (6.6%) in 2018 (1,2). Triple‑negative BC (TNBC) often signature of EMT. Thus, suppressing the expression of N‑Ca could potentially decrease the invasion and migration of cancer cells (14). Vimentin is one of the most widely expressed and highly conserved proteins in the type III intermediate fila‑ ment protein family (15), and is known to promote the invasion Correspondence to: Ms. Min Li or Professor Fangfang Zhang, and migration of cancer cells. Furthermore, EMT markers Department of Pathology, School of Basic Medical Sciences, are associated with the biological features of tumors, such as Lanzhou University, 222 South Tianshui Road, Lanzhou, the characteristics of the tumor, and its invasive and migra‑ Gansu 730000, P.R. China tory abilities. Detection of changes in the expression levels of E‑mail: [email protected] E‑mail: [email protected] EMT markers could assist in investigating the differences in the biological features and the changes in protein expression *Contributed equally levels. This method has been used to evaluate the prognosis of patients with hepatocellular carcinoma, prostate cancer and Key words: coatomer protein complex subunit β2, triple‑negative BC (8,16,17). breast cancer, migration, invasion, phosphorylated‑AKT The coatomer protein complex subunit β2 (COPB2) gene located in chromosome 3q2.3 encodes the 102‑kDa nucleoprotein COPB2, which is a member of the 7 protein 2 WU et al: SILENCING THE COPB2 GENE Golgi coatomer complex. COPB2 is essential for budding vimentin (cat. no. 5741S; Cell Signaling Technology, Inc.), and vesicular trafficking between the endoplasmic reticulum p‑AKT (cat. no. 4060T; Cell Signaling Technology, Inc.), and Golgi membrane. It is therefore essential in maintaining AKT (cat. no. 4685S; Cell Signaling Technology, Inc.) and cellular homeostasis, including the transcriptional regulation AKT agonist SC79 (cat. no. HY‑18749; MedChemExpress). and signal transduction of cells (18‑22). GAPDH (cat. no. YM3040; ImmunoWay Biotechnology Previous studies have revealed that the expression of Company) served as the protein loading control and was probed COPB2 is significantly upregulated in prostate cancer, chol‑ using a monoclonal antibody. ImageJ 1.51j8 software (National angiocarcinoma, lung cancer and colon cancer cells, and that Institutes of Health) was used to analyze the density of the bands. it enhances proliferation (23‑26). Furthermore, COPB2 regu‑ lates the proliferation of colon cancer cells via the JNK/c‑Jun MTT cell proliferation assay. HS‑578T and MCF‑7 cells were signaling pathway (26). Moreover, silencing the COPB2 gene seeded at a cell density of 2x103 cells/well in 96‑well plates and decreases the expression levels of proteins associated with the cultured at 37˚C with 5% CO2. Cell densities were analyzed receptor‑tyrosine kinase (RTK) signaling pathway in gastric on day 1, 2, 3, 4 and 5 after seeding. At each time point, the cancer cells, such as EGFR, HER‑2, fms related RTK 3 and cells were incubated with 20 µl MTT solution for 4 h at 37˚C, phosphorylated (p)‑AKT (27). and then cells were treated with 150 µl DMSO with constant The role of COPB2 in the pathogenesis of TNBC remains agitation. After a 10‑min incubation at room temperature, poorly understood. However, circumstantial evidence reported the supernatant was collected and MTT signals at an optical in the abovementioned literature suggests that COPB2 may be density (OD) of 490 nm were quantitatively detected using a a key contributor to the phenotype of TNBC cells and that its microplate reader (Bio‑Rad Laboratories, Inc.). action may be mediated via the AKT signaling pathway. The present study hypothesized that the increased expression level Colony formation assay. HS‑578T and MCF‑7 cells were of COPB2 in TNBC cells may contribute to the occurrence of seeded in 6‑well plates at a density of 1x103 cells/well and EMT, which could promote the migration and invasion capaci‑ cultured at 37˚C with 5% CO2 for 10 days. Cells were then ties of TNBC cells in vitro. This effect may be mediated via stained for 10 min with crystal violet and fixed with 4% para‑ the AKT signaling pathway. formaldehyde for 30 min at room temperature. The number of cell colonies was counted, and images were captured under a Materials and methods light microscope. Cell lines and culture conditions. The clonal TNBC HS‑578T Cellular migration and invasion assay. Cell migration and inva‑ and non‑TNBC MCF‑7 cell lines (The Type Culture Collection sion assays were performed using a 24‑well Transwell chamber of The Chinese Academy of Sciences) were cultured at 37˚C (pore size, 8 µm; Corning, Inc.) as previously described (26). with 5% CO2 in DMEM (Hyclone; Cytiva) supplemented with For the invasion assay, the upper chambers were coated with 10 and 15% FBS (Biological Industries). 40 µl Matrigel (BD Biosciences) diluted in DMEM for 30 min at 37˚C. HS‑578T and MCF‑7 cells were cultured in the upper Lentiviral transduction of HS‑578T cells. HS‑578T cells were chamber at a density of 2x105 cells/ml (200 or 160 µl/well seeded in 6‑well culture plates (4x104 cells/well) and incubated for migration and invasion assays, respectively). The upper at 37˚C until 30% confluent. Cells were then divided into three chambers were submerged into the lower chamber containing groups: Infection with lentiviral vector (Lv)‑short hairpin 15% FBS‑supplemented medium (500 µl). After 24 and 48 h (sh)RNA COPB2 with a green fluorescent protein (GFP) tag in culture at 37˚C (5% CO2), non‑migrated cells that remained (sh‑COPB2 from Shanghai GenePharma Co., Ltd.); infection in the upper chambers were removed using a cotton swab. with empty lentiviruses with a GFP tag (sh‑Control; Shanghai The migrated or invaded cells on the opposite side of the GenePharma Co., Ltd.); or no infection. sh‑COPB2 (AGA membrane were stained with 0.1% crystal violet for 10 min TTA GAG TGT TCA ATT A) was inserted into Gv248 lentiviral at room temperature, and counted under an ECLIPSE 80i vectors (Shanghai GenePharma Co., Ltd.) and the cells were fluorescence microscope. infected as previously described (26). Then, 96 h after the final infection, the cells were analyzed for infection efficiency by Statistical analysis. The dataset regarding COPB2 gene counting the number of GFP‑positive cells under a fluores‑ expression and histological grade of BC was obtained from cence microscope (ECLIPSE 80i; Nikon Corporation). The Cancer Genome Atlas (TCGA) database. The differen‑ tial expression analysis was performed using the R package, Immunoblots.