COPB2 is up‐regulated in breast cancer and plays a vital role in the metastasis via N‐cadherin and Vimentin

Abstract Breast cancer (BC) is a common malignant tumour for the adult female and its relative incidence has increased continuously in recent years. The primary molecular mechanisms of breast tumourigenesis remain unclear. With the sequencing technology, we found that coatomer protein complex subunit beta 2 (COPB2) gene is overexpressed in breast cancer tissues. However, the biological function of COPB2 in BC has yet to be determined. This current research demonstrates, significant up‐regulation of COPB2 in tissues of breast cancer while comparing the adjacent normal tissue both invalidated cohort and TCGA cohort. Up‐regulated expression of COPB2 was correlated with lymph node metastasis (LNM) and oestrogen receptor (ER) in the TCGA cohort and a high level of COPB2 was associated with age and lymph node metastasis in the validated cohort. Besides, logistic analysis illustrated in BC patient COPB2 expression, tumour size, age, ER and disease stage were independent high‐risk factors of LNM. Loss of function experiments revealed that down‐regulation of COPB2 could inhibit capacities of proliferation and cell invasion in MDA‐MB‐231 and BT‐549 cell lines. Moreover, underexpression of COPB2 could decrease the EMT‐related protein N‐cadherin and vimentin which may lead to cell invasion. This current research provides new shreds of evidence that COPB2 overexpression shows significant character in the progression of breast cancer. To best of our knowledge, our findings indicated that COPB2 was vital oncogene which was associated with breast cancer.


| INTRODUC TI ON
Breast cancer (BC) is one of the widespread malignant cancer in women with 268 670 newly projected identified cases and 41,400 expected deaths in the United States in 2018. 1 Significant improvement has been made in recent decades and breast cancer can be cured with surgery, endocrine therapy, cytotoxic or targeted therapies. Throughout the past years, emerging research has been done in discovering the underlying mechanisms of BC. [2][3][4] However, the therapeutic effect is not satisfactory. [5][6][7] Due to the development of medical technology and the perfection of social welfare, part of the breast cancer patient can be treated by surgery, endocrine therapy, chemotherapy or targeted therapies. However, the effect of those treatments is not always gratifying. 8,9 Oestrogen receptor (ER)-positive (or luminal) tumours represent around 60% to 75% of all breast cancers and endocrine therapy is highly effective and appropriate for nearly all females with tumours.
Unfortunately, some subtype of breast cancers often becomes resistant to endocrine treatment, relapse and becomes fatal, underscoring the need for the identification of novel molecular biomarkers that can predict the progression and prognosis of this disease.
The coatomer protein complex subunit beta 2 (COPB2, also known as Beta-Cop, P102 or Coatomer Protein Complex Subunit Beta Prime) gene encodes a 102-kDa protein and maps to chromosome 3q23. COPB2 is a subunit of the Golgi coatomer complex, a cytosolic protein complex that composes the coat of non-clathrincoated vesicles and is crucial for vesicular trafficking and Golgi budding. Recently, COPB2 is involved in tumourigenesis in numerous kinds of cancer including lung adenocarcinoma and prostate cancer. 10,11 However, the clinical significance of COPB2 in patients with breast cancer remains unknown.
In this study, we performed quantitative real-time PCR (qRT-PCR) in 56 breast cancer tissues and its adjacent normal tissue to validate the results of sequencing technology. Subsequently, we used the small interfering RNA (siRNA) to knockdown the expression of COPB2 in two types of breast cancer cell lines (MDA-MB-231 and BT-549). Furthermore, the expression of COPB2 in breast cancer tissues and adjacent non-cancerous tissues was both analysed in a TCGA cohort and local cohort. In the TCGA cohort, logistic analysis illustrated in BC patient COPB2 expression, tumour size, age, oestrogen receptor and disease stage were independent high-risk factors of LNM.
Our study aimed to determine the relationship between COPB2 expression and the role of COPB2 in the proliferation and metastasis in breast carcinoma.

| Patients and samples
Fifty-six breast cancer tissues and 56 paired adjacent non-cancerous tissues were gained from the Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University.
Samples were frozen in liquid nitrogen immediately after surgical removal and stored at −80°C until further RNA detection. All patient's information was collected under the protocols approved by the institutional review board of the Ethics Committee of the First Affiliated Hospital of Wenzhou Medical University (approval no. 2012-57).

| RNA extraction and real-time quantitative polymerase chain reaction (RT-qPCR)
According to the manufacturer's instructions (Invitrogen, USA), RNA from cells was isolated by TRIZOL reagent (Invitrogen, USA) according to the manufacturer's protocol. The purity of RNA was examined at 260/280 nm by spectrophotometry (Thermo, San Jose, CA, USA).
All RNA samples were reverse transcribed (Toyobo, Osaka, Japan). The comparative CT method (∆∆CT) method was used to evaluate the relative quantification of COPB2. Each sample was performed in triplicate.

| Cell invasion and migration assay
To detect cell capacity of migration and invasion, we used cell cul- Then, the chamber was placed in the incubator in the above environment (37°C with 5% CO 2 ). After 24 hours, the chamber was carefully removed and fixed with 4% paraformaldehyde and 0.4% crystal violet was used for staining the membrane in 15 minutes.
Five random fields of view were selected for analysis and images were captured by a microscope at 20 × magnification. All images were captured by the microscope camera. Above experiments were performed in triplicate.

| Apoptosis detection analysis
Annexin-V-FITC apoptosis kit (Nanjing KeyGen Biotech. Co., Ltd., Nanjing, China) was used to determine the proportion of apoptotic cells. We collected the transfected cells in 6-plate and centrifuged

| Protein extraction and Western blot analysis
The collected cells were cleaved in cell lysis buffer (Beyotime, China) to obtain whole-cell lysates. Protein concentrations were measured using bicinchoninic acid assay (BCA). We used 10% SDS-PAGE on the gel to separate total cell lysate proteins. Then all samples were electro-transferred onto PVDF membranes. For blocking the blots, the TBST mixed 5% non-fat milk was used for 2 hours at room temperature. Next, the blots were probed with polyclonal antibody at 4°C overnight. Finally, the blots were incubated with the secondary antibody (goat anti-rabbit IgG conjugated with HRP, Abcam, CA) for 1 hour at room temperature.

| COPB2 was significantly up-regulated in breast cancer
Through the whole transcriptome sequencing of 15 pairs of the breast tumour and adjacent normal tissues, we found that the expression level of COPB2 in breast cancer tissues was significantly higher than that in normal tissues (Table 1). To validate the result of sequencing, we selected another 56 paired breast cancer tissues and adjacent non-cancerous tissues to examine the expression of COPB2 mRNA by qRT-PCR. As shown in Figure 1, the expression of COPB2 mRNA expression was significantly up-regulated in breast cancer tissues compared with the adjacent normal tissues (P < 0.001). In order to further investigate the dysregulated expression of COPB2,  Figure 2A, P < 0.001). These results suggested that COPB2 gene may act as a potential tumour oncogene in breast cancer patients.

| The relationship between COPB2 expression and clinicopathologic characteristics in breast cancer
To investigate whether COPB2 was associated with the development and progression of breast cancer, a total of 1091 breast cancer patients were divided into low and high COPB2 expression groups according to the median value. As shown in Table 2, the human oestrogen receptor (ER) expression (P = 0.038) and lymph node metastasis (P = 0.035) were significantly related to the expression of COPB2.
However, there were no differences between the two groups in terms of tumour size, age, distant metastasis, clinical stage and progesterone receptor (PR). In our validated cohort, we found that higher COPB2 expression group had more lymph node metastasis (P = 0.033) and younger age (P = 0.007) than lower COPB2 expression group (Table 3).

| High expression levels of COPB2 were correlated with LNM in breast cancer
In order to assess the effects of COPB2 expression on the prognosis of breast cancer, we analysed follow-up data of the TCGA cohort.
As showed in Table 4

| COPB2 gene knockdown inhibits breast cancer cell proliferation and promotes apoptosis
Considering COPB2 was overexpressed in above BC cell lines, we hy-

| Down-regulation of COPB2 inhibits breast cancer cell migration and invasion
In order to validate the function of COPB2 in the breast cancer cell, we performed the cell migration and invasion assay. As expected, underexpression of COPB2 can significantly inhibit breast cancer cell's capacities of migration and invasion compared with the si-NC control group (Figure 5A-D).

| The COPB2 gene regulates epithelialmesenchymal transition (EMT)
Accumulating researches have increasingly recognized epithelialmesenchymal transition (EMT) as a vital process during cancer cell metastasis. [12][13][14] As shown in Figure 6, the expression of vimentin and N-cadherin was deceased in si-COPB2 cell lines. Those result suggested that COPB2 may promote BC cell metastasis by influencing EMT.

| D ISCUSS I ON
Breast cancer is the most frequent malignant cancers which are diagnosed and it is also one of the main reasons for death worldwide. 1,14 In China, the occurrence rate of breast cancer has soared obviously in recent decades. 15  Being a heterogeneous disease, the information provided by the hormone receptor status is always not enough for proper treatment. 16 Although much progress in breast cancer research has been made, the specific molecular mechanisms of breast cancer remain unknown. High-throughput sequencing analysis of genetic alterations, an advanced sequencing method, have been utilized to explore its molecular mechanisms. COPA and COPB have the amino acid repeat of tryptophan and aspartic acid that has been connected to up-regulation of the cell cycle, signal transduction as well as apoptosis. 20   In spite of our interesting discovery, there are still some limitations to the study.
First, more tumour samples are needed to analyse the correlation of COPB2 with clinical parameters and confirm its role in BC metastasis. Besides, in vivo experiments and precise cellular mechanisms for COPB2 are required to be further studied.

| CON CLUS IONS
In short, our research shows the increased expression of COPB2 in breast cancer in humans when compared to normal tissues and COPB2 may predict breast cancer metastasis and also it might be an independent molecular marker.

ACK N OWLED G M ENTS
The authors would like to thank all the doctors of Department of Thyroid and Breast Surgery, The First Affiliated Hospital of Wenzhou Medical University, (Wenzhou, China) for providing all the necessary information required for this study. Ouchen Wang and Duping Huang designed the whole work.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
Ethical approval for this study was obtained from the Ethics

Committee of the First Affiliated Hospital of Wenzhou Medical
University.

CO N S E NT FO R PU B LI C ATI O N
Written informed consent was issued by the patients for the publication of this research and accompanying images. A copy of the written consent is ready for review by the Editor in Chief of this journal.
Consent for publication was obtained from all participants.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets supporting the conclusions of this study are included in this article and its, additional images. Raw data are available on the main electronic data storage system of First Affiliated Hospital of Wenzhou Medical University and access can be provided upon request to the authors.