Long non‐coding RNA ZEB2‐AS1 promotes the proliferation, metastasis and epithelial mesenchymal transition in triple‐negative breast cancer by epigenetically activating ZEB2

Abstract The triple‐negative breast cancer is the most malignant type of breast cancer. Its pathogenesis and prognosis remain poor despite the significant advances in breast cancer diagnosis and therapy. Meanwhile, long noncoding RNAs (LncRNAs) play a pivotal role in the progression of malignant tumors. In this study, we found that LncRNA‐ZEB2‐AS1 was dramatically up‐regulated in our breast cancer specimens and cells (MDA231), especially in metastatic tumor specimens and highly invasive cells, and high lncRNA‐ZEB2‐AS1 expression is associated with clinicopathologic features and short survival of breast cancer patients. LncRNA‐ZEB2‐AS1 promotes the proliferation and metastasis of MDA231 cells in SCID mice. Thus, it is regarded as an oncogene in triple‐negative breast cancer. It is mainly endo‐nuclear and situated near ZEB2, positively regulating ZEB2 expression and activating the epithelial mesenchymal transition via the PI3K/Akt/GSK3β/Zeb2 signaling pathway. Meanwhile, EGF‐induced F‐actin polymerization in MDA231 cells can be suppressed by reducing lncRNA‐ZEB2‐AS1 expression. The migration and invasion of triple‐negative breast cancer can be altered through cytoskeleton rearrangement. In summary, we demonstrated that lncRNA‐ZEB2‐AS1 is an important factor affecting the development of triple‐negative breast cancer and thus a potential oncogene target.

is the most in clinical treatment, and it is also a hot and difficult issue of current research.
Many previous studies confirmed that epithelial mesenchymal transition (EMT) is one of the main mechanisms that cause the dispersion of malignant tumors. 3 EMT triggers a variety of biological changes in normal mammary epithelial cells, which eventually obtains the characteristics of mesenchymal cells. This effect enhances the metastasis capacity, invasiveness and resistance of cancer cells, thereby preventing apoptosis and promoting the production of extracellular matrix components. 4 The occurrence of EMT is related to a variety of molecular mechanisms and signaling pathways. 5 Its main function is the reduction of intercellular adhesion and E-cadherin, mesenchymal vimentin, and N-cadherin expression. Moreover, many transcriptional factors, including snail and ZEB, have crucial roles in EMT-induced processes. 6 The ZEB family (ZEB1 and ZEB2) is closely related to the EMT-related markers of malignant tumors. 7 However, the regulatory role of lncRNA-ZEB2-AS1 in ZEB2 expression in breast cancer remains unreported.
LncRNA is a kind of regulatory RNA that has a transcriptional length of more than 200 nucleotides and has no protein coding ability. 8 LncRNA plays a vital role in malignant tumor development, especially in apoptosis, proliferation, and invasion. [9][10][11] Abnormal ln-cRNA expression is observed in malignant tumors, such as gastric cancer, hepatocellular carcinoma (HCC), and glioma. [12][13][14] Moreover, lncRNA is a competitive endogenous RNA (ceRNAs) that regulates miRNA. 15,16 Despite the significant role lncRNA in malignant tumor development, its regulatory functions and molecular mechanisms remain poorly understood.
In this study, we found that abnormal lncRNA-ZEB2-AS1 expression is associated with patient survival and prognosis and controls ZEB2 expression. In the MDA231 cells, LncRNA-ZEB2-AS1 promoted the proliferation and metastasis of tumor cells and triggered EMT via the PI3K/Akt/GSK3β/Zeb2 signaling pathway and F-actin polymerization.

| Clinical specimens
We obtained breast cancer specimens (BC) and adjacent no tumor (ANT) specimens from the Affiliated Hospital of Weifang Medical University from 2011 to 2016. All the specimens were frozen to −150°C. None of the specimens received radiotherapy or chemotherapy before surgery. Informed consent was obtained from each patient. The study was approved by the Research Ethics Committee of Weifang Medical University.

| Plasmid construction and cell transfection
MDA231 cells (2 × 10 5 ) were planted in six-well plates and left overnight. We used Lipofectamine 2000 to transfect the MDA231 cells in accordance with the manufacturer protocol Lnc-ZEB2-AS1-RNAi, and si-NC plasmids were constructed by GeneChem company (shanghai, China) Sequences were provided in Table S1.
Stable transfected cells were maintained with 300 μg/mL G418 for 14 days.

| Wound healing assay
Approximately 2 × 10 5 breast cancer cells were planted onto six-well plates and then left overnight. Wounds were created with a 10 μL pipette tip. Complete medium including 1% FBS was added to the plates with or without EGF. The entire setup was photographed after 36 hours.

| Cell proliferation assays
For the cell counting kit 8 (CCK-8; Solarbio, Beijing) assays, approximately 2 × 10 3 cells were seeded in a 96-well plate. The proliferation capacities of the cells cultured for 24,48,72,96, and 120 hours were tested by the CCK-8 assays. The cell growth curves were plotted by using the absorbance value at each time point.

| Western blot
Protein expression was examined as described previously. 17 The following antibodies were employed: ZEB2 (Santa Cruz,

| Quantitative real-time PCR
We performed Quantitative real-time PCR (QRT-PCR) to define the relative level of lncRNA-ZEB2-AS1. GAPDH levels were used for normalization. We used TRIzol to extract RNA from fresh breast cancer specimens and cells. Then, cDNA was synthesized with the total RNA by using an M-MLV reverse transcriptase kit (Promega, USA). Relative mRNA expression was normalized through the 2 − ΔΔCT method. QRT-PCR was carried out using an Applied Biosystems 7500.

| Cellular F-actin measurement
MDA231 cells were immobilized with 4% paraformaldehyde with PBS. The cells were then washed three times before they were blocked with a buffer containing goat serum for 45 minutes. The MDA231 cells were stained with Phalloidin (Fluorescein isothiocyanate) FITC for 1 hours, washed three times, and covered with fluorescence decay resistant sealing tablets. Data processing was performed as described previously. 18

| Immunofluorescence
The MDA231 cells were fixed with 4% paraformaldehyde for 25 min.
The cells were permeabilized with 0.1% Triton X-100 and blocked with goat serum for 45 min at 37°C. The primary antibodies of relation were incubated at 4°C overnight. FITC, Cy3-labeled goat antirabbit IgG secondary antibodies, and DAPI were used.

| Animal studies
The Server Combined Immune-deficiency (SCID) mice were provided by the Animal Care and Use Committee of Wei Fang Medical University. Si-NC/MDA231 and MDA231/si-ZEB2-AS1 cells (2 × 10 6 ) were injected into the oxter of each female SCID mouse (n = 10). When the xenografts became evident, tumor volume was measured. After 7 weeks, metastasis in the lung tissues was examined by HE staining.

| Statistical analysis
Data were analyzed by SPSS v16.0. All values were expressed as mean ± SD. The results were analyzed with T test or ANOVA. Χ 2 -test was used for the analysis of the connection between lncRNA-ZEB2-AS1 and the clinicopathologic features. P < 0.05 was considered significant in all cases

| Up-regulation of lncRNA-ZEB2-AS1 in breast cancer specimens and cells was related to clinicopathologic features and decreased survival of breast cancer patients
To determine the biological function of lncRNA-ZEB2-AS1 in the tumorigenesis of triple-negative breast cancer, we detected lncRNA-ZEB2-AS1 expression levels in 98 paired of BC specimens and ANT specimens by qRT-PCR. The results revealed that lncRNA-ZEB2-AS1 was more markedly up-regulated in breast cancer specimens than paired ANTs ( Figure 1A). We further detected expressed lncRNA-ZEB2-AS1 with or without lymph node metastasis in breast cancer specimens ( Figure 1B up-regulated in breast cancer cells compared with MCF-10A cells.
The expression levels of highly invasive MDA231 cells and MDA435 were higher lncRNA-ZEB2-AS1, and those of invasive cells T47D and MCF-7 were lower than lncRNA-ZEB2-AS1 ( Figure 1C). LncRNA is considered to have different biological functions in different locations of the cells. Meanwhile, we first detected the distribution of lncRNA-ZEB2-AS1 in the MDA231 cells. LncRNA-ZEB2-AS1 was mainly located in the nucleus was minimally expressed in the cytoplasm ( Figure 1D). Additionally, breast cancer patients with high lncRNA-ZEB2-AS1 levels have shorter survival rates than those with low levels ( Figure 1E). The relationship between lncRNA-ZEB2-AS1 expression level and the clinicopathologic features of breast cancer patients was evaluated. LncRNA-ZEB2-AS1 expression is highly associated with tumor differentiation, lymph node metastasis, and distant metastasis in breast cancer but not associated with age or tumor size ( Table 1). The results indicated that lncRNA-ZEB2-AS1 plays an oncogenic role and is associated with clinicopathologic features and decreased survival of breast cancer patients.

| Knockdown of lncRNA-ZEB2-AS1 suppressed the proliferation and invasion of MDA231 cells
The knockdown of lncRNA-ZEB2-AS1 in breast cancer cells implies that lncRNA-ZEB2-AS1 have important roles in breast cancer progression. To further investigate whether lncRNA-ZEB2-AS1 is connected to the occurrence of breast cancer, we researched the function of lncRNA-ZEB2-AS1 in vitro. As shown in Figure 2A Figure 3D).

| Knockdown of lncRNA-ZEB2-AS1 led to the down-regulation of ZEB2 expression through PI3K/Akt/GSK3β/Zeb2 signaling pathway and inhibited EMT
The upstream antisense transcription may affect corresponding gene expression. 19 LncRNA-ZEB2-AS1 is an antisense lncRNA. We  stimulation. The data showed that EGF led to short actin polymerization in si-NC/MDA231 cells and was notably decreased in the MDA231/si-ZEB2-AS1 cells, indicating that lncRNA-ZEB2-AS1 played a vital function in modulating cytoskeleton rearrangement by EGF stimulation ( Figure 5A,B). Meanwhile, LIMK and cofilin are two vital proteins related to the regulation of F-actin polymerization. 20 We detected the phosphorylation status of LIMK and cofilin, to confirm whether lncRNA-ZEB2-AS1 inhibits EGF-induced rearrangement of cytoskeletons in the MDA231 cells ( Figure 5C). As shown in Figure 5C, the reduction of lncRNA-ZEB2-AS1 expression depressed the phosphorylation levels of LIMK and cofilin, and the cytoskeleton was abolished. These results indicated that reduction of lncRNA-ZEB2-AS1

| Clinical relevance of lncRNA-ZEB2-AS1 between ZEB2 and EMT related markers in human breast cancer
In order to further indicate the clinically relevant by which lncRNA-ZEB2-AS1 modulated ZEB2 and EMT related markers. We detected the lncRNA-ZEB2-AS1 expression and its association with the expression levels of ZEB2 in the clinical tissues of breast cancer paired with breast cancer specimens and ANT specimens. The relationship between the expression levels of EMT related markers and lncRNA-ZEB2-AS1 was investigated. In Figure 6, a positive correlation among lncRNA-ZEB2-AS1, ZEB2, and vimentin in 30 tested clinical specimens was observed, whereas lncRNA-ZEB2-AS1 was negatively correlated with E-cadherin. All the results suggested that lncRNA-ZEB2-AS1 in the ZEB2 and EMT-related markers is clinically relevant in breast cancer.

| D ISCUSS I ON
An increasing number of studies have reported that LncRNA considerably affects cellular processes, such invasion, metastasis, metabolism, and apoptosis. 21,22 LncRNAs monitor carcinogenesis principally via modulating the expression levels of oncogenes or tumor suppressors. 19 Many studies revealed that LncRNA-ZEB2-AS1 decreases tumor growth and metastasis in HCC. 23 LncRNA-ZEB2-AS1 is a newly discovered lncRNA, whose clinical meaning, biological roles, and explicit mechanism are poorly understood in breast cancer. We first confirmed that lncRNA-ZEB2-AS1 is abnormally expressed in breast cancer specimens, and this abnormality is related Collectively, our results showed that lncRNA-ZEB2-AS1/PI3K/ Akt/GSK3β/Zeb2 axis facilitates tumor progression and is a potential prevention target in breast cancer.

CO N FLI C T O F I NTE R E S T
The authors confirm that there are no conflicts of interest.