LncRNA ZEB1‐AS1 down‐regulation suppresses the proliferation and invasion by inhibiting ZEB1 expression in oesophageal squamous cell carcinoma

Abstract Multiple studies have unveiled that long non‐coding RNAs (lncRNAs) play a pivotal role in tumour progression and metastasis. However, the biological role of lncRNA ZEB1‐AS1 in oesophageal squamous cell carcinoma (ESCC) remains under investigation, and thus, the current study was to investigate the functions of ZEB1‐AS1 in proliferation and invasion of ESCC. Here, we discovered that ZEB1‐AS1 and ZEB1 were markedly up‐regulated in ESCC tissues and cells relative to their corresponding normal control. ZEB1‐AS1 and ZEB1 overexpressions were both related to TNM staging and lymph node metastasis as well as poor prognosis in ESCC. The hypomethylation of ZEB1‐AS1 promoter triggered ZEB1‐AS1 overexpression in ESCC tissues and cells. In addition, ZEB1‐AS1 knockdown mediated by siRNA markedly suppressed the proliferation and invasion in vitro in EC9706 and TE1 cells, which was similar with ZEB1 siRNA treatment, coupled with EMT alterations including the up‐regulation of E‐cadherin level as well as the down‐regulation of N‐cadherin and vimentin levels. Notably, ZEB1‐AS1 depletion dramatically down‐regulated ZEB1 expression in EC9706 and TE1 cells, and ZEB1 overexpression obviously reversed the inhibitory effects of proliferation and invasion triggered by ZEB1‐AS1 siRNA. ZEB1‐AS1 shRNA evidently inhibited tumour growth and weight, whereas ZEB1 elevation partly recovered the tumour growth in ESCC EC9706 and TE1 xenografted nude mice. In conclusion, ZEB1‐AS1 overexpression is tightly involved in the development and progression of ESCC, and it exerts the antitumour efficacy by regulating ZEB1 level in ESCC.

chemotherapy, radiotherapy and surgery are still main therapy strategies for patients with ESCA, but the therapy efficacy is not very satisfactory, which may be because of the facts that most patients with ESCA was diagnosed in an advanced stage, coupled with the appearance of metastasis loci. 10,11 Therefore, it is in dire need of seeking for the new therapeutic strategy to improve the patients' prognosis.
Long non-coding RNA (lncRNA) is a group of transcripts longer than 200 nucleotides, which can be split into 6 different types as follows: promoter-associated transcripts, sense, antisense, bidirectional, intronic, intergenic and 3′UTR-associated transcripts. 12,13 LncRNAs may be located in the cytoplasm or nucleus, but are mainly present in cell nucleus. 14 LncRNAs are widely involved in the regulation of diverse biological processes. 15,16 In recent years, increasing evidence has revealed the important regulatory roles of lncRNAs in the occurrence and progression of ESCA. 17 Notably, lncRNA-miRNA-mRNA regulatory axis widely participates in oesophageal carcinogenesis. 18 Many lncRNAs play pivotal roles in maintaining and promoting the biological characteristics of tumour cells, and thus, lncRNAs may be the attractive therapeutic targets in a variety of tumours. 19,20 Our recent work identified many differential lncRNA through TCGA database, and we revealed that ZEB1-AS1 was significantly up-regulated in ESCA, 21 but its precise functions remain unknown. It is a fact that ZEB1-AS1 is closely correlated with tumour occurrence and development, such as bladder cancer, 22 glioma, 23 melanoma 24 and non-small-cell lung cancer, 25 and these data suggest that ZEB1-AS1 is involved in tumour progression via multiple different molecular mechanisms, suggesting the complexity of ZEB1-AS1 function in these tumours. However, how ZEB1-AS1 is regulated during ESCC development is still unclear, and therefore, herein, the expression pattern of ZEB1-AS1 and its regulatory role in the proliferation and invasion ability of ESCC were investigated, which will propose ZEB1-AS1/ZEB1 regulatory axis as an underlying therapeutic target for ESCC therapy.

| Tissue samples
Resected ESCC tissues and normal oesophageal epithelial tissues were collected from the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China, including 56 ESCC samples and 56 paired normal samples, which was stored in liquid nitrogen.
Informed consent of all tissue samples confirmed by pathologist was obtained from each participant. All patients did not receive any treatments prior surgery. The current study was authorized by the Institutional Research Ethics Committee of Zhengzhou University.
ISH assay for ZEB1-AS1 expression was carried out according to previous report. 26 In brief, tissue sections were deparaffinized in xylene and graded alcohol, followed by heat treatment for 15 minutes in the buffer at 100°C. The tissues were treated using pepsin for 10 minutes at room temperature (RT), and then, ZEB1-AS1 probe labelled with digoxigenin was added to the tissue sections. The tissue slides were denatured 96°C for 5 minutes and were placed in a moisturized chamber for hybridization reaction overnight at 37°C. Finally, NBT/ BCIP was employed to develop the signal. PBS was used as a negative control instead of ZEB1-AS1 probe.

| Immunohistochemistry (IHC)
Immunohistochemistry assay was carried out according to previous document. 27 Briefly, tumour tissues with paraffin embedding were serially cut with 4 μm, and the sections were dewaxed and rehydrated according to standard protocol. Subsequently, 3% H 2 O 2 was employed to inactivate the endogenous peroxidase for 10 minutes, followed by heating the tissue sections in EDTA solution (1 mmol/L, pH 9.0) for antigen retrieval. The sections were rinsed using PBS buffer for 5 minutes and then blocked using 3% BSA solution for 30 minutes at RT. Afterwards, the sections were incubated with ZEB1 primary antibody (Abcam) with 1:200 dilution overnight at 4°C, and then, horseradish peroxidase (HRP)-conjugated secondary antibody was added to sections for 30 minutes at RT. The staining signal was developed with a DAB kit (Zhongshan Golden Bridge Biotechnology Company).

| Staining scores of ISH and IHC
All staining results were independently evaluated in a double-blinded manner by two pathologists according to the following standards.

| Methylation-specific PCR (MSP)
Methylation-specific PCR was performed according to previous report. 28 The primer for MSP was designed using MethPrimer online software as described previously in the following 28

| Western blot
Total proteins were extracted from ESCC cells using RIPA lysis (Solarbio), and the concentration was measured by Bradford method.
The proteins were separated by SDS-PAGE and then transferred to PVDF membranes (Millipore Corporation). The primary antibodies against E-cadherin, N-cadherin, vimentin, ZEB1 and β-actin (1:200 dilution, Abcam) were incubated with PVDF membrane (Roche) overnight at RT after blocking with skimmed milk. Subsequently, the secondary antibody (ZSGB-BIO) was added to PVDF membrane.
Finally, enhanced chemiluminescence reagents (Beyotime) were utilized to develop the protein signal.

| Animal experiment
Female BALB/c nude mice with 4-6 weeks old were purchased from Weitong Lihua Experimental Animal Technical Company. All mice were maintained in a pathogen-free facility. EC9706 and TE1 cells (1 × 10 6 ) harbouring stable ZEB1-AS1 knockdown were subcutaneously inoculated into the back of nude mice. Once the tumour volumes reached approximate 100 mm 3 , the mice were randomly split into three groups: pLVX-shRNA-NC, pLVX-shRNA-ZEB1-AS1 and pLVX-shRNA-ZEB1-AS1 plus ZEB1. Tumour volumes were measured twice a week.
When the measurement was terminated, tumour growth curve was made. All protocols were approved by the Institutional Committee for Use and Care of Laboratory Animals of Zhengzhou University.

| Statistical assay
All data expressed as mean ± standard deviation (SD) were repeated in triplicate, which were examined using GraphPad Prism 6.0 software. The data regarding ISH and IHC were examined using chisquare, and survival assay was performed using log-rank test. The comparisons of two groups were determined using t test, and comparisons of three groups or above were investigated using one-way ANOVA. A P value less than 0.05 were regarded to be significant.

| ZEB1-AS1 and ZEB1 levels in ESCC tissues and cells and their prognosis power in ESCC
TCGA database integrating UALCAN and starBase was employed to investigate the ZEB1-AS1 and ZEB1 levels in ESCC tissues and its prognostic value. We unveiled that the levels of ZEB1-AS1 and ZEB1 were up-regulated in ESCA tissues ( Figure 1A,B), and their expressions displayed markedly positive correlations in ESCA tissues ( Figure 1C).
Notably, ZEB1-AS1 was not related to the prognosis of the patients with ESCA ( Figure 1D), but the survival ratio of the patients with high ZEB1 level in different grade ESCA patients was lower than that with low ZEB1 level (P < .05) ( Figure 1E). To validate these findings, qPCR, ISH and IHC were utilized to detect the levels of ZEB1-AS1 and ZEB1 in 56 cases of ESCC tissues and para-carcinoma tissues. The results of ISH and IHC demonstrated that ZEB1-AS1 and ZEB1 expressions in ESCC tissues (positive ratio: 44.6% and 41.1%) were both higher than those in normal tissues (14.3% and 12.5%) (Figure 2A-D), which were also confirmed by qPCR ( Figure 2E,F). Our results herein imply that ZEB1-AS1 and ZEB1 may play oncogenic role in ESCC.

| The correlations of ZEB1-AS1 and ZEB1 expressions with clinicopathological features in ESCC
To explore the possible biological role of ZEB1-AS1 and ZEB1 in ESCC, SPSS 21.0 software was utilized to dissect the correlations of ZEB1-AS1 and ZEB1 levels with clinicopathological factors, respectively. The results revealed that ZEB1-AS1 and ZEB1 levels were both associated with lymph node metastasis and TNM staging (P < .01), but not correlated with patients' gender, age, invasion depth and histological grade (P > .05) ( Table 1 and 2). These data imply that ZEB1-AS1 and ZEB1 may exert pivotal role in the development and progression of ESCC.

| ZEB1-AS1 and ZEB1 are both correlated with TNM staging, lymph node metastasis and poor prognosis in ESCC
To further explore the underlying role of ZEB1-AS1 and ZEB1 in TNM staging, lymph node metastasis and prognosis in ESCC, qRT-PCR was used to analyse the associations of ZEB1-AS1 and ZEB1 with TNM staging, lymph node metastasis and prognosis in ESCC. We found that ZEB1-AS1 levels in ESCC patients with III + IV staging and lymph node metastasis were markedly higher than those with I + II staging and without lymph node metastasis ( Figure 3A Figure 4C). The results from different oesophageal cells revealed that methylation level of ZEB1-AS1 promoter in different ESCC cells was evidently lower than that in Het-1A cell ( Figure 4D). These data imply that ZEB1-AS1 at high level may be tightly associated with ZEB1-AS1 promoter hypomethylation.

| ZEB1-AS1 down-regulation suppresses proliferation and invasion ability in ESCC
To preliminarily dissect ZEB1-AS1 functions in ESCC, the effects of ZEB1-AS1 siRNA on cell proliferation and invasion ability of ESCC F I G U R E 2 The expressions of ZEB1-AS1 and ZEB1 in ESCC tissues and paired normal tissues. A, In situ hybridization detection for ZEB1-AS1 level in normal oesophageal epithelial tissues, bar = 100 μm; B, In situ hybridization detection for ZEB1-AS1 level in ESCC tissues, bar = 100 μm; C, Immunohistochemistry assay for ZEB1 protein expression in normal oesophageal epithelial tissues, bar = 100 μm; D, Immunohistochemistry assay for ZEB1 protein expression in ESCC tissues, bar = 100 μm; E, qPCR detection for ZEB1-AS1 level in ESCC tissues and paired normal tissues; F, qPCR detection for ZEB1 mRNA level in ESCC tissues and paired normal tissues

| ZEB1 siRNA markedly suppresses the proliferation and invasion ability in ESCC
To further elucidate whether ZEB1 also exerts a pivotal role in proliferation and invasion of ESCC, we detected the effect of ZEB1 siRNA on proliferation and invasion ability in ESCC cells. F I G U R E 5 ZEB1-AS1 down-regulation contributed to the inhibition of cell proliferation and invasion in ESCC cells. A, qPCR detection of ZEB1-AS1 expression in EC9706 and TE1 cells after transfection with si-NC and si-ZEB1-AS1; B and C, CCK-8 assay for cell proliferation after transfection with si-NC and si-ZEB1-AS1-2, * P < .05 and ** P < .01, compared with si-NC group; D, transwell chamber assay for cell invasion after transfection with si-NC and si-ZEB1-AS1-2; E and F, statistical assay for invasive cell number in EC9706 and TE1 cells, ** P < .01, compared with si-NC group; G, Western blot assay for the expressions of E-cadherin, N-cadherin and vimentin proteins in different treatment EC9706 and TE1 cells; H and I, the relative expression of E-cadherin, N-cadherin and vimentin proteins in different treatment EC9706 and TE1 cells, ** P < .01, *** P < .001 and **** P < .0001, compared with si-NC group We found ZEB1 siRNA significantly suppressed ZEB1 expression in EC9706 and TE1 cells ( Figure 6A,B). Further CCK-8 experiment revealed that ZEB1 down-regulation contributed to proliferation inhibition in EC9706 and TE1 cells ( Figure 6C,D). Besides, ZEB1 down-regulation markedly inhibited invasion ability in EC9706 and TE1 cells (Figure 6E,F,G). Our data indicate that ZEB1 may exert the crucial regulatory role in cell proliferation and invasion in ESCC cells.
F I G U R E 6 ZEB1 down-regulation elicited the inhibition of proliferation and invasion in ESCC cells. A, ZEB1 siRNA markedly reduced ZEB1 protein expression in EC9706 and TE1 cells; B, relative level of ZEB1 protein in EC9706 and TE1 cells, **** P < .0001, compared with si-NC group; C, CCK-8 experiment assay for cell proliferation in si-NC group and si-ZEB1 group in EC9706 cells, * P < .05 and ** P < .01, compared with si-NC group; D, CCK-8 experiment assay for cell proliferation in si-NC group and si-ZEB1 group in TE1 cells, * P < .05 and ** P < .01, compared with si-NC group; E, transwell chamber investigation for cell invasion ability in EC9706 and TE1 cells; F, statistical assay for invasive cell number in si-NC group and si-ZEB1 group in EC9706 cells, ** P < .01, compared with si-NC group; G, statistical assay for invasive cell number in si-NC group and si-ZEB1 group in TE1 cells, ** P < .01, compared with si-NC group F I G U R E 7 ZEB1-AS1 suppresses cell proliferation and invasion by targeting ZEB1. A, ZEB1 protein level was evaluated by Western blot after transfection with si-ZEB1-AS1 in EC9706 and TE1 cells; B, Relative level of ZEB1 protein after transfection with si-ZEB1-AS1 in EC9706 and TE1 cells, *** P < .001 and **** P < .0001, compared with si-NC group; C: CCK-8 experiment assay for cell proliferation in si-NC group, si-ZEB1-AS1 and si-ZEB1-AS1 plus ZEB1 overexpression group in EC9706 cells, ** P < .01, compared with si-NC group; D: CCK-8 experiment assay for cell proliferation in si-NC group, si-ZEB1-AS1 and si-ZEB1-AS1 plus ZEB1 overexpression group in TE1 cells, ** P < .01, compared with si-NC group; E, transwell chamber assay for cell invasion ability in si-NC group, si-ZEB1-AS1 and si-ZEB1-AS1 plus ZEB1 overexpression group in EC9706 and TE1 cells; F, statistical assay for invasive cell number in si-NC group, si-ZEB1-AS1 and si-ZEB1-AS1 plus ZEB1 overexpression group in EC9706 cells, *** P < .001 and **** P < .0001, compared with si-NC group; G, statistical assay for invasive cell number in si-NC group, si-ZEB1-AS1 and si-ZEB1-AS1 plus ZEB1 overexpression group in TE1 cells, *** P < .001, compared with si-NC group

| ZEB1 overexpression reverses the inhibitory effect of proliferation and invasion mediated by ZEB1-AS1 siRNA in ESCC cells
It is well documented that antisense lncRNAs control the level of the sense genes directly or indirectly. 29 ZEB1-AS1 is an antisense cognate gene of ZEB1, and we put forward that whether ZEB1-AS1 can regulate ZEB1 expression, whereas whether ZEB1 overexpression can reverse the biological process mediated by ZEB1-AS1 downregulation in ESCC cells. Therefore, the effect of ZEB1-AS1 on ZEB1 expression and the biological effect triggered by ZEB1 overexpression were detected. The results revealed that si-ZEB1-AS1 markedly down-regulated ZEB1 protein level in EC9706 and TE1 cells ( Figure 7A,B). Further investigation showed that si-ZEB1-AS1 strikingly restrained cell proliferation and invasion ability, whereas ZEB1 overexpression obviously reversed the inhibitory effect of proliferation and invasion elicited by si-ZEB1-AS1 ( Figure 7C-G), suggesting ZEB1-AS1 plays a vital regulatory role in cell proliferation and invasion by manipulating ZEB1 expression in ESCC cells.

| ZEB1-AS1 down-regulation suppresses tumour growth in ESCC cell xenografted nude mice
To explore the potential role of ZEB1-AS1 in tumorigenesis in ESCC and TE1 cells xenografted nude mice, which was partly reversed by ZEB1 overexpression (Figure 8E,F). Our data herein imply that ZEB1-AS1 suppresses tumour growth by inhibiting ZEB1 level in ESCC.

| D ISCUSS I ON
In the current study, ZEB1-AS1 functioned as a regulator of ZEB1 The data from meta-analysis revealed that high ZEB1-AS1 level was closely correlated with overall survival (HR = 2.16, 95% CI: 1.89-2.47), poor histological grade, high tumour staging and lymph node metastasis among patients with cancer, 36 which was similar with previous report. 37 These findings indicate that suppression of ZEB1-AS1 expression may be a potential strategy for therapy of many tumour patients. Here, we found ZEB1-AS1 and ZEB1 expressions in ESCC tissues were both higher than those in normal tissues, and their high levels were both associated with TNM staging as well as lymph node metastasis (P < .01). More importantly, high ZEB1-AS1 and ZEB1 F I G U R E 8 ZEB1-AS1 down-regulation suppresses tumour growth in EC9706 and TE1 xenografted nude mice. A, ZEB1-AS1 downregulation suppressed tumour growth in EC9706 xenografted nude mice, whereas ZEB1 overexpression partly reversed the inhibitory efficacy, ns indicates no significance, ** P < .01, compared with pLVX-shRNA-NC group and pLVX-shRNA-ZEB1-AS1 plus ZEB1; B, Tumour weight in different treatment group, **** P < .0001, compared with pLVX-shRNA-NC group and pLVX-shRNA-ZEB1-AS1 plus ZEB1; C, ZEB1-AS1 down-regulation suppression tumour growth in TE1 xenografted nude mice, whereas ZEB1 overexpression partly reversed the inhibitory efficacy, ns indicates no significance, ** P < .01, compared with pLVX-shRNA-NC group and pLVX-shRNA-ZEB1-AS1 plus ZEB1; D, Tumour weight in different treatment group, **** P < .0001, compared with pLVX-shRNA-NC group and pLVX-shRNA-ZEB1-AS1 plus ZEB1; E, Western blot assay for E-cadherin, N-cadherin and vimentin protein experssions in EC9706 and TE1 xenografted nude mice, and β-actin was used for loading control; F, relative levels of E-cadherin, N-cadherin and vimentin protein experssions in EC9706 and TE1 xenografted nude mice, **** P < .0001, compared with pLVX-shRNA-ZEB1-AS1 levels both predicted poor prognosis of patients with ESCC. These data suggest that ZEB1-AS1 and ZEB1 may participate in the progression and metastasis of ESCC. To elucidate the underlying factors regarding ZEB1-AS1 overexpression in ESCC, the methylation status of ZEB1-AS1 promoter in ESCC tissues and cells was detected in this study. We found that methylation level of ZEB1-AS1 promoter in ESCC tissues and cells was significantly lower than that in normal tissues and cells (P < .0001), and meanwhile, a negative correlation between methylation level of ZEB1-AS1 promoter and ZEB1-AS1 expression was found, which is consistent with previous investigation in hepatocellular carcinoma. 28 These data imply that ZEB1-AS1 at high level may be tightly associated with ZEB1-AS1 promoter hypomethylation.
Sustaining proliferative signalling and activating invasion and metastasis have been verified to be two main characteristics of tumours, 38 and combined targeting two hallmarks may be a novel strategy for therapy of tumour patients. Cheng R et al revealed that ZEB1-AS1 depletion suppressed the growth, migration, invasion and EMT in cervical cancer, which may be achieved by inhibiting ZEB1 expression. 39 Moreover, ZEB1-AS1 depletion markedly restrained the proliferation and induced apoptosis in colorectal cancer, whereas ZEB1-AS1 elevation elicited the opposite effect. 40

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

AUTH O R CO NTR I B UTI O N
Yan Zhao and Shujun Yang: designed the whole experiment, dissected the data and prepared for the manuscript. Yan Zhao, Ning Wang, Xiaoshan Zhang and Hongtao Liu: performed the experiment, obtained the data and reviewed the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.