Clinical significance of long non‐coding RNA ZEB2‐AS1 and EMT‐related markers in ductal and lobular breast cancer

Abstract Background Breast cancer is considered the most prevalent type of cancer in women and accounts for a high rate of death. A body of research has demonstrated that lncRNAs have a regulatory function in human diseases, especially cancers. ZEB2‐AS1 is known as an oncogenic lncRNA in various types of cancers, and its deregulation may contribute to cancer development and progression. Therefore, we aimed to reveal the association of ZEB2‐AS1 expression with epithelial–mesenchymal transition (EMT) markers, as a hallmark of cancer progression, in a clinical setting. Methods A recent study suggested that ZEB2‐AS1 is significantly involved in EMT. Here we intended to explore the roles of lncRNA ZEB2‐AS1 in breast cancer (BC) using bioinformatics tools and laboratory settings. We first evaluated the expression of ZEB2‐AS1 mRNA in tumor and healthy control tissues by lnCAR database. Furthermore, ZEB2‐AS1 expression level, ZEB2, E‐cadherin, and vimentin was measured via qRT‐PCR in 30 paired ductal and lobular carcinoma tissues from breast cancer patients and the normal adjacent ones. The correlation between the lncRNA ZEB2‐AS1 expression and clinicopathological characteristics of the breast cancer patients was evaluated. Results ZEB2‐AS1 showed an upregulation in breast cancer tissues (p = .04) compared to normal adjacent samples. In addition, its level was higher in breast cancer patients with advanced Stages (III & IV) (n = 18) compared to early Stages (I & II) (n = 12) (p = .04). Moreover, ZEB2 (p = .01) and vimentin (p = .02) expression were upregulated in the BC sample, but the expression level of E‐cadherin (p = .02) was downregulated when compared with the adjacent normal tissues. By comparison of the expression of EMT‐markers between different stages of breast cancer, overexpression of ZEB2 (p = .04) and vimentin (p = .04) and down expression of E‐cadherin (p = .03) was observed in advance stages. Conclusions Collectively, our findings suggest that ZEB2‐AS1 expression is significantly upregulated in tumor tissues, especially in advanced stages and ZEB2‐AS1 is associated with the aggressiveness of tumors by functioning as putative oncogenic lncRNA. In addition, a combination of ZEB2‐AS1 and these EMT markers in breast cancer potentiates these genes as biomarkers for tumor progression.

lncRNA. In addition, a combination of ZEB2-AS1 and these EMT markers in breast cancer potentiates these genes as biomarkers for tumor progression.

K E Y W O R D S
biomarker, breast cancer, EMT, LncRNA ZEB2-AS1, non-coding RNA

| INTRODUCTION
Breast cancer (BC) is among the highly prevalent causes of mortality in women. Despite advanced diagnostic and therapeutic methods, the prognostic attempts for this type of cancer remain disappointing. 1 Breast cancer cells are invasive and often have distant metastasis. 2 Metastasis is one of the causes of treatment failure. Hence, comprehension of the mechanism of cancer progression can be useful in managing breast cancer. 1 The long non-coding RNA (lncRNA) is a member of non-proteincoding RNAs, the length of which is more than 200 nt. LncRNAs are capable of regulating gene expression at the transcriptional and posttranscriptional levels. LncRNAs have a functional role in malignancy development, mainly in migration and invasion. 3,4 Several lines of studies have evidenced the aberrant expression and functional deregulation of lncRNAs in various malignancies including pancreatic cancer and hepatocellular carcinoma (HCC). It has been reported that lncRNA dysfunction might be significantly contributing to the development of breast cancer. 5 For example, LncRNA DANCR, 6 and LncRNA NEAT1 7 contribute to tumor progression; by elevating proliferation, migration, and epithelial-mesenchymal transition (EMT) in breast cancer.
EMT is considered a mechanism that causes the progression and migration of tumors. Epithelial cells obtain specifications of mesenchymal cells via EMT. In EMT, we observe deregulation in cell-cell junctional proteins such as E-cadherin (downregulation), vimentin and N-cadherin (upregulation). 8 ZEB2-AS1 is a natural antisense transcript of the 5 0 UTR of zinc finger e-box binding homologous box 2 (ZEB2). Studies indicated that ZEB2-AS1 promotes cancer progression in multiple tumor types including bladder cancer, 9 gastric cancer, 10

| Expression profile analysis
The research obtained the ZEB2-AS1 expression pattern in ductal breast cancer and adjacent normal tissue using datasets provided by the lnCAR database (https://lncar.renlab.org/). 13 lnCAR is a universal database that precisely displays differential expression profiles and prognostic landscape in human cancers by re-annotating microarray probes. This server provides expression analysis for 54 893 samples across 10 cancers based on microarray from the GEO database.

| Overall survival analysis
The GEPIA2 (http://gepia2.cancer-pku.cn/#survival) accomplishes survival analyses using the expression levels of the gene or isoform. 14 The prognostic effects of gene expression level can be checked by users and the large TCGA and GTEx datasets can be searched too.
The GEPIA2 web tool was employed to test the correlation of ZEB2-AS1, the overall survival (OS), and Relapse-Free Survival (RFS) in BC patients. In addition, we applied the survival map segment that compares the survival contribution of ZEB2-AS1, ZEB2, vimentin, and E-cadherin in OS and RFS of BC patients, calculated via hazard ratio (HR) based on the Mantel-Cox test based on their gene expression analysis and survival time (Month). We considered levels (75%) and (25%) as the expression threshold for classifying high and low expression categories, respectively. The marked box around the tiles indicates statistical significance (p < .05) in cancer types. Poor and good prognoses are indicated by red and blue colors, and the color intensity indicates the HR level.

| In silico analysis of correlation
Besides, we assessed correlation analysis between ZEB2-AS1 and EMT-related markers ZEB2, Vimentin, and E-cadherin using StarBase 3.0 online server. StarBase 3.0 (http://starbase.sysu.edu.cn/) is an interactive web resource for large-scale analysis of the RNA-RNA and protein-RNA interplay networks and can be used for correlation analysis between two genes (mRNAs, lncRNAs, miRNAs, and pseudogenes) in multiple cancer types. 15

| Clinical specimens
We obtained 30 pairs of ductal and lobular carcinoma BC tissues (12 ER + , 9 PR + , and 9 HER2 + ) as well as adjacent normal tissues from the Emam Khomeini Hospital affiliated with the Tehran University of Medical Science. We used liquid nitrogen to freeze all the fresh specimens. All patients signed the written informed letter of consent. The patients had not received the treatment previously, and carcinoma was approved by pathological diagnostic tools by expert pathologists who resided in the hospital. This project was confirmed by the

| RNA extraction and qRT-PCR
We isolated the total RNA from tissue specimens utilizing the TRIzol reagent (GeneAll, Korea) according to the manufacturer's protocol.
The RNA samples were then reversely transcribed to cDNA using a PrimeScript RT reagent kit (Takara, Japan). Next, we measured the gene expression via a quantitative real-time polymerase chain reaction (qRT-PCR) with the SYBR-Green Master Mix (Ampliqon, Denmark). Each sample was performed in triplicate. We considered GAPDH as the endogenous control. Gene expression levels were normalized to GAPDH by using the 2 ÀΔΔct method. Table 2 enlists the primer sequences.

| Statistical analysis
For data analysis, GraphPad Prism 8 was used. The difference between groups was analyzed using Student's t-test (differences between 2 groups) and ANOVA (among several groups). A p-value under .05 was interpreted as significant in all data. For depicted receiver operating characteristic (ROC) curve, we used SPSS software (IBM SPSS statistics 26). The combination ROC was obtained using regression logistics and enter model.

| ZEB2-AS1 expression level in BC tissues
A previous study has reported that ZEB2-AS1 correlated positively with the progression of many tumors. 16 To assess the ZEB2-AS1 T A B L E 1 Clinical information of patient samples. T A B L E 2 Primer sequence.
The expression profile of the ZEB2-AS1 in breast cancer. The expression of ZEB2-AS1 was determined in tumor versus normal tissues of BC by the lnCAR database. p < .05 was considered statistically significant.
The relative expression of the ZEB2-AS1 in tumor compared with matched normal tissues (A). The ZEB2-AS1 mRNA level between cancer tissues with early-stage and advanced-stage samples (B) and differentially expressed lncRNA-ZEB2-AS1 in breast cancer specimens with and without lymph node metastasis (C). p < .05 was considered statistically significant.
T A B L E 3 Result of association between genes expression and patient's features.  Furthermore, the correlation of ZEB2-AS1 expression level was analyzed with patients' clinicopathological variables (Table 3). Overexpression of ZEB2-AS1 was notably related to advanced stages of breast cancer (p = .04; Figure 2B) and lymph node metastasis (p = .03; Figure 2C). We could not find a correlation between the expression of ZEB2-AS1 with the rest of the clinicopathological features.

| In silico analysis of overall survival
We conducted a survival analysis using the ZEB2-AS1 expression pat-

| Expression level of ZEB2 and EMT-induce markers in BC tissues
According to previous investigations, we found that ZEB2-AS1 is an antisense lncRNA that is transcribed antisense from the ZEB2 gene. 17 ZEB2 acts as a transcription factor that can modulate E-cadherin and F I G U R E 3 Survival curve evaluating the prognostic value of ZEB2-AS1. Overall Survival curves have been plotted for patients with BC using the GEPIA2. A log-rank p < .05 was considered statistically significant. F I G U R E 4 Survival analysis of ZEB2-AS1, ZEB2, vimentin, and E-cadherin in BC via gene expression analysis and survival duration based on TCGA data. High-expression (75%) and low-expression (25%) were considered as the expression threshold for splitting two (high vs. low) patient groups with BC. The highlighted box around the tiles displays statistical significance ( p < .05). Red and blue colors had a poor and good prognosis, respectively, and the strength of the color identified the HR value.

| Correlation of ZEB2-AS1 with ZEB2 and EMT markers
We evaluated the clinical correlation of ZEB2-AS1 transcriptional levels with ZEB2 and EMT markers in BC tissues using the qRT-PCR technique. The result indicated that ZEB2-AS1 was positively correlated with ZEB2 and vimentin, while a negative association was observed between ZEB2-AS1 and E-cadherin ( Figure 8). Taken together, our findings revealed that ZEB2-AS1 expression level was associated with vimentin, but this correlation was not significant with ZEB2 and E-cadherin in BC patients (Table 4).

| ROC curve analysis
We plotted the ROC curve using the true positive fraction (sensitivity) versus false-positive fraction (1-specificity) across different cutoff F I G U R E 8 Clinical relevance of lncRNA-ZEB2-AS1 between ZEB2 and EMT-related markers in human breast cancer. Correlation between lncRNA-ZEB2-AS1 and ZEB2 (A) and correlation between lncRNA-ZEB2-AS1 and vimentin (B) and correlation between lncRNA-ZEB2-AS1 and Ecadherin (C). p < .05 was considered statistically significant.
T A B L E 4 Correlation between ZEB2-AS1 with EMT-related marker. F I G U R E 9 The diagnostic power of ZEB2 and ZEB2-AS1 expression in BC by using ROC curve analysis. ROC curve analysis with respect to the ZEB2 gene expression in distinguishing between the early stage and the final stage of breast cancer (A). ROC curve analysis with respect to lncRNA-ZEB2-AS1 was implemented to discriminate between tumor and normal tissues of breast cancer (B). Combined evaluating the diagnostic power of ZEB2-AS1, ZEB2, vimentin, and E-cadherin gene expression in determining tumor and normal tissues in breast cancer employing CombiROC analysis. A considerable enhancement in AUC (95%), as well as enhanced sensitivity and specificity, was observed. p < .05 was considered statistically significant (C).
points. The diagnostic capacity of genes was evaluated by computation of the area under the curve (AUC) estimates. Among the four genes under study, ZEB2 showed the best diagnostic potential to discriminate between the early stage and end-stage (AUC value: 82%) ( Figure 9A). The AUC value for lncRNA ZEB2-AS1 was 71% ( Figure 9B).
Moreover, the combination of ZEB2-AS1, ZEB2, vimentin, and E-Cadherin increased the AUC to 95%. The results of the ROC curve analysis are shown in Figure 9C. Moreover, the elevated expression was associated with a higher stage and lymph node metastasis (lnCAR database does not provide information on ZEB2-AS1 differential expression between BC stages).
One study in triple-negative breast cancer determined increased ZEB2-AS1 expression in patients with lymph node metastasis compared with patients without lymph node metastasis. 11 This study accordance with our results but we performed the present study on different samples that were ER-positive or PR-positive.
In order to examine the survival analysis, we used the GEPIA2 Past studies have shown that EMT is the procedure in that epithelial cells lose their polarity and cellular connections and acquire mesenchymal properties. 28 In addition, dysregulation in EMT-markers expression occurs in the cell, leading to EMT and tumor metastasis. The TGF-β pathway is a cellular pathway involved in the development of EMT in cancers. Activation of the TGF-β pathway increases the EMTmarker transcription such as Snail1 and ZEB2. 29 Snail is a transcription factor that stimulates the transcription of lncRNA ZEB2-AS1. 30 LncRNA ZEB2-AS1 is a lncRNA that is transcribed towards antisense from ZEB2 and can regulate the ZEB2 gene via binding to ZEB2 mRNA and prevent the removal of an intron in 5 0 -UTR. The present ZEB2 intron is actually an internal ribosome entry site (IRES) to increase the translation of this gene. As a pivotal transcription factor, ZEB2 may serve a tumor progression function in cancers, especially in EMT. 12 Increased ZEB2 expression inhibits transcription of E-Cadherin and promotes vimentin expression, which is a mesenchymal factor. with EMT-related markers in head and neck squamous cell carcinoma (HNSCC) and triple-negative BC and showed a positive association between ZEB2-AS1 and ZEB2/vimentin. In addition, these studies illustrated that ZEB2-AS1 has a negative correlation with E-cadherin. 11,31 In the present research, a correlation study of ZEB2-AS1 and EMT-related markers indicated that ZEB2-AS1 has a positive correlation with ZEB2 and vimentin, whereas there is a negative correlation between ZEB2-AS1 and E-cadherin, but this association was not statically significant (ZEB2; E-cadherin), which might be due to the insufficient sample size. The ROC curve analysis suggested a diagnostic potential for the ZEB2 gene in discriminating the stages of cancer and probably metastasis in breast cancer. In addition, combination ROC indicated that these genes (ZEB2-AS1, ZEB2, vimentin, and E-cadherin) could have the potential to diagnostic of breast cancer metastasis. However, more research is needed to confirm this finding. We had limitations in collecting samples, and due to ZEB2-AS1 being a recently discovered lncRNA, there was little information about this in a variety of databases.

| CONCLUSION
In conclusion, our findings showed that lncRNA ZEB2-AS1 upregulation could act on breast cancer onset and development through ZEB2, vimentin, and E-cadherin dysregulation. This lncRNA probably has a diagnostic and prognostic characterization in breast cancer metastasis and it seems to be a good therapeutic and pharmaceutics value to prevent breast cancer progression. Therefore, these four genes may be used in putative diagnostic panels to differentiate the malignant condition and metastasis of breast tissues. Although, more functional studies are required to validate our findings.