Down‐regulation of long non‐coding RNA HOTAIR inhibits invasion and migration of oesophageal cancer cells via up‐regulation of microRNA‐204

Abstract Oesophageal cancer is a progressive tumour with high mortality. However, therapies aimed at treating oesophageal cancer remain relatively limited. Accumulating studies have highlighted long non‐coding RNA (lncRNA) HOX transcript antisense RNA (HOTAIR), microRNA‐204 (miR‐204) and homeobox C8 (HOXC8) in the progression of oesophageal cancer. Herein, we tried to demonstrate the function of HOTAIR, miR‐204 and HOXC8 in oesophageal cancer and their relationship. Differentially expressed genes involved in oesophageal cancer were identified. The endogenous expression of HOTAIR and miR‐204 in oesophageal cancer cell lines was altered to elucidate their effects and to identify the interaction among HOTAIR, miR‐204 and HOXC8. We also explored the underlying regulatory mechanisms of HOTAIR and miR‐204 with siRNA against HOTAIR, miR‐204 mimic or miR‐204 inhibitor. Cell proliferation, migration, invasion and apoptosis were subsequently detected. Xenograft in nude mice was induced to evaluate tumourigenicity. miR‐204 was down‐regulated, while HOTAIR and HOXC8 were up‐regulated in the oesophageal cancer tissues. HOTAIR could competitively bind to miR‐204 and miR‐204 could further target HOXC8. The oesophageal cancer cells treated with si‐HOTAIR or miR‐204 mimic exhibited decreased expression levels of HOXC8, Vimentin and MMP‐9, but increased E‐cadherin level. Silenced HOTAIR or elevated miR‐204 inhibited proliferation, migration and invasion, along with stimulated apoptosis of oesophageal cancer cells. In summary, our results show that lncRNA HOTAIR could specifically bind to miR‐204 as a competing endogenous RNA and regulate miR‐204 and HOXC8. Hence, down‐regulation of HOTAIR could inhibit progression of oesophageal cancer, indicating a novel target for oesophageal cancer treatment.


| INTRODUC TI ON
Oesophageal cancer, an aggressive cancer, has been reported to be the sixth most deadly cancer globally. 1 There are two major histological types of oesophageal cancer: squamous cell carcinoma and adenocarcinoma, 2 and they both show a typical syndrome known as dysphagia, which negatively affect the quality of life of the patients. 3 Oesophageal cancer is often accompanied by a high mortality mainly due to the low rate of early diagnosis, consequently resulting in a poor prognosis, 4,5 although a pre-operative chemoradiotherapy has been reported to be able to improve the survival rate of the patients who have potentially curable oesophageal or oesophagogastricjunction cancer. 6 Existing literature has implicated long non-coding RNAs (lncRNAs) in the occurrence and development of numerous aggressive tumours. 7,8 Thus, the current study aimed to investigate the role of lncRNAs might play in the progression of oesophageal cancer.
Long non-coding RNAs are a family of RNAs that have no coding capacity however still exhibit the ability to play a crucial role in various biological regulatory processes 9 such as a performance as a competing endogenous RNA (ceRNA) influencing post-transcriptional regulation by interfering the pathways of microRNA (miRNA or miR). 10 A previous study revealed the diagnostic potential of serum lncRNA HOX transcript antisense RNA (HOTAIR) as a promising biomarker for oesophageal squamous cell carcinoma. 11 HOTAIR is a 2148-nucleotide-long lncRNA that has been shown to participate in the development of the physiological epidermis as well as the progression of cancer, and potentially as a regulator of tumour suppressor genes. 12 Apart from lncRNA, accumulating evidence has highlighted the crucial inhibitory role of miRs in blocking the development of oesophageal cancer, such as microRNA-204 (miR-204), which performs as a tumour suppressor. [13][14][15][16] HOTAIR has been reported to influence the progression of oesophageal squamous cell carcinoma by binding to endogenous miR-125 and miR-143. 17 Besides, studies have also suggested that HOTAIR regulates HOX genes, 18,19 whose expression has been detected in oesophageal cancer cell lines, including HOXC8. 20 Homeobox C8 (HOXC8) is a transcription factor capable of stimulating oncogenes in various malignancies, and it is implicated in the modulation of multiple proteins that linked with cancer. 21 Although the aforementioned literature has highlighted a relationship between HOTAIR, miR-204 and HOXC8, their functions in the development of oesophageal cancer remain unknown.
Hence, the current study aimed to investigate its underlying molecular mechanism.

| Ethics statement
The current study was performed with the approval of the ethics committee of The Linyi People's Hospital. All participants signed informed consents. All animal experiments were conducted in strict accordance with the Guide for the Care and Use of Laboratory Animal by International Committees.

| Microarray-based gene expression profiling
Gene expression data of oesophageal cancer were downloaded from the Cancer Genome Atlas (TCGA) (http://cance rgeno me.nih.gov/) database. Differential analysis was conducted in order to analyse the transcriptome profiling data using the R package "edgeR" 22 False positive discovery (FDR) correction was performed based on P-value with package "multitest". FDR < 0.05 and |log2 (fold change)| >2 was set as the threshold for screening the differentially expressed genes (DEGs). The lncRNA-binding miRNA candidates were identified from the miRcode website (http://www.mirco de.org/), and the target genes of miRNAs were predicted based on the miRTarBae website (http://mirta rbase.mbc.nctu.edu.tw/ php/index.php). According to the oesophageal cancer staging criteria developed by the Union for International Cancer Control (UICC), 23 18 patients were at the T1 or T2 stage, and 28 patients were at the T3 or T4 stage. 31 patients had lymph node metastasis (LNM) while the remaining did not. Meanwhile, adjacent normal tissues (normal mucous tissues ≥10 cm from oesophageal cancer tissues) from all enrolled participants were obtained immediately following surgery and regarded as the control group. The collected tissues were stored at −80°C prior to use.

| Immunohistochemistry
The collected oesophageal cancer tissues were fixed in 4% paraformaldehyde for 24 hours, dehydrated by 80%, 90% and 100% ethanol and n-butyl alcohol, then immersed in a 60°C wax box, embedded and sliced into 5 μm sections for immunohistochemistry assay. The sections were subsequently baked at 60°C for 1 hour and then dewaxed by xylene, followed by dehydration with gradient alcohol, immersion in 3% H 2 O 2 for 10 minutes and wash with distilled water. After high-pressure antigen retrieval for 90 seconds, the sections were cooled to room temperature and washed three times with phosphate buffer saline (PBS), 3

| Cell line selection
Five oesophageal cancer cell lines, namely EC8712, EC8733, ECA109, EC9706 and EC8501 as well as normal oesophageal epithelial cell line HEEC (Shanghai Institute of Biochemistry and Cell Biology, Shanghai, China) were cultured in the RPMI 1640 medium containing 10% serum at 37°C with 5% CO 2 . The medium was replaced every 2-3 days based on cell growth. The cells were passaged when they covered 80%-90% of the culture plates. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed in order to select the cell lines with the highest HOTAIR and miR-204 expression for further experimentation.

| Dual luciferase reporter gene assay
The interaction site between miR-204 and HOTAIR or HOXC8 was predicted by the biological prediction website (https ://cm.jeffe rson. edu/rna22/ ) and the binding sequences were obtained to construct luciferase-tag plasmids. The full length of HOTAIR and the 3'untranslated region (3'UTR) of HOXC8 were transferred, respectively, to the pmirGLO luciferase vector (E1330, Promega Corporation, Madison, WI) via clonal amplification and referred to as pHOTAIR-Wt and pHOXC8-Wt. The mutations were introduced on the binding site based on the sequence of pHOTAIR-Wt and pHOXC8-Wt with the resulting constructs referred to as pHOTAIR-Mut and pHOXC8-Mut, respectively. The pRL-TK vector (E2241, Promega Corporation, Madison, WI) expressing renilla luciferase was considered as the internal control. Next, miR-204 mimic and miR-204 negative control (NC) were respectively co-transfected with luciferase reporter vector into HEK-293T, after which luminance was detected using a luminometer (Glomax20/20, Promega Corporation, Madison, WI).

| RNA pull-down assay
The cells were transfected with 50 nmol/L biotin-labelled WT-bio-miR-204 and MUT-bio-miR-204. At 48 hours post-transfection, the cells were collected, washed with PBS and then incubated in the specific lysate buffer (Ambion, Austin, Texas) for 10 minutes. The lysates were further incubated with M-280 streptavidin beads (S3762, Sigma-Aldrich St. Louis, MO) that had been pre-coated with RNasefree BSA and yeast tRNA (TRNABAK-RO, Sigma-Aldrich St. Louis, MO). Following a 3 hours period of incubation at 4°C, the beads were washed twice with pre-cooled lysate buffer, thrice with lowsalt buffer and once with high-salt buffer. The combined RNA was purified using Trizol, after which HOTAIR was detected by RT-qPCR.

| RNA-immunoprecipitation assay
The cells were treated with lysis buffer (25 mmol/L Tris-HCl pH = 7.4, 150 mmol/L NaCl, 0.5% NP-40, 2 mmol/L ethylenediamine tetraacetic acid (EDTA), 1 mmol/L NaF and 0.5 mmol/L dithiothreitol) containing RNasin (Takara, Dalian, Liaoning, China) and protease inhibitors (B14001a, Roche, Basel, Switzerland). The lysate was Trizol method was performed to obtain RNA from the beads, after which the expression of HOTAIR was determined by RT-qPCR. tion had been removed. The next day, the cells were washed three times with cleaning Lotion I (5 minutes per wash) in order to reduce the background signal, followed by washing with lotion II and once more with lotion III at 42°C under conditions void of light. Next, 4',6diamidino-2-phenylindole (DAPI) was employed to stain the cells for 10 minutes, which followed by three PBS washes at room temperature. The coverslips with migrated cells were subsequently carefully removed from the wells under dark conditions, fixed and then mounted with a medium for fluorescence detection. HOTAIR specific probe was synthesized by Ribo Biotech Co., Ltd., (Guangzhou, Guangdong, China). The cells were washed with serum-free medium and then incubated with the complex for 6-8 hours at 37°C with 5% CO 2 , after which the medium was replaced with a complete culture medium.  Table 1). The cDNA was subjected to fluorescence qPCR according to the instructions of the SYBR ® Premix Ex TaqTM II kit (TaKaRa, Dalian, Liaoning, China). ABI7500 fluorescence qPCR (ABI Company, Oyster Bay, NY) was employed in order to perform RT-qPCR with GAPDH and U6 regarded as the internal controls. The expression pattern of HOTAIR, miR-204, HOXC8, E-cadherin, Vimentin and MMP-9 was subsequently determined. Each experiment was repeated independently three times.

| Western blot analysis
The cells were initially washed three times with pre-cooled PBS, following a 48 hours period of transfection. After additional PBS washing, the cells were incubated with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime Biotechnology Co., Shanghai, China) in a 1.5 mL centrifugation tube. After, centrifugation at 14 000 g for 10 minutes and collection of the supernatant, the protein concentration was determined using the bicinchoninic acid (BCA) method, after which the supernatant was stored at −20°C. The collected proteins were subjected to electrophoresis separation using 10% separation gel and 5% spacer gel that were prepared using a sodium dodecyl sulfate-polyacrylamide gel electrophoresis kit. The separated proteins were transferred to the nitrocellulose membrane via the wet method, and blocked with 5% BSA at room temperature

| Scratch test
Following 48 hours of transfection, the transfected cells in each group were seeded in a 6-well plate (5 × 10 5 cells/well). In the event of cell confluence reaching approximately 90%, a scratch was made using a sterile pipette along the central axis across the well. Cells failing to adhere to the wall were removed by PBS washing, with serumfree culture medium subsequently added for an additional 0.5-1-h culture to induce cell recovery. The cells were photographed at 0 and 24 hours after cell recovery. The Image-Pro Plus Analysis software (Version X; Media Cybernetics, Silver Springs, MD) was employed to measure cell migration distance. The relative migration rate was calculated with the blank group regarded as the control based on the following formula: relative migration rate = (migration distance experimental group /migration distance blank group ) × 100% (the control value was 1 or 100%). The experiment was independently repeated three times. Matrigel was considered to be a reflection of the invasion ability.

| Transwell assay
Each experiment was independently repeated three times.

| Tumour xenografts in nude mice
A total of 66 clean Kunming nude mice (age: 4-6 weeks, weight:

| Statistical analysis
All experimental data were analysed using SPSS 21.0 statistical software (IBM Corp., Armonk, NY). Measurement data were expressed as mean ± SD. Kolmogorov-Smirnov was employed to assess normal distribution. Data of oesophageal cancer tissues and adjacent normal tissues that conformed to normal distribution were analysed by paired t test and those that conformed to skewed distribution were analysed by the non-parametric Wilcoxon signed-ranks test. Data from multiple groups were compared by one-way ANOVA. Pairwise comparisons between mean values were analysed by least significant difference (LSD) and cell viability or tumour volume at different time-points was compared by repeated measurement ANOVA.
A P < 0.05 was considered to be statistically significant.

| miR-204 is down-regulated while HOTAIR and HOXC8 are up-regulated in oesophageal cancer tissues
Gene expression microarray analysis was performed to screen differentially expressed lncRNAs, miRNAs and genes associated with (IL-11) ( Figure 1D). Considering the mechanism by which IL-11 influences cancer has been thoroughly studied, 25,26 we aimed to investigate the mechanism by which HOTAIR acting as a ceRNA of miR-204 regulates the expression of HOXC8 in oesophageal cancer cells.
Immunohistochemistry and RT-qPCR were conducted in order to determine the expression pattern of HOXC8, HOTAIR and miR-204 in the oesophageal cancer tissues as well as the adjacent normal tissues. The immunohistochemistry ( Figure 1E,F) and RT-qPCR ( Figure 1G)

| miR-204 binds to HOTAIR and HOXC8
Data provided by online software, suggested the existence of bind- pull-down assay revealed that the relative HOTAIR enrichment was significantly increased in WT-miR-204 when compared with MUT-miR-204 (P < 0.05), indicating that miR-204 could directly bind to HOTAIR ( Figure 2E). The results of the Ago2 RIP assay, revealed that compared with IgG, HOTAIR enrichment was significantly increased in Ago2 (P < 0.05), suggesting that HOTAIR could directly bind to Ago2 protein ( Figure 2F). The aforementioned results indicated that HOTAIR could participate in the regulation of HOXC8 by competitively binding to miR-204. The FISH results exhibited that HOTAIR was predominately distributed in the cytoplasm ( Figure 2G).

| EC9706 and ECA109 cell lines were selected for experiments
Several cell lines were evaluated in order to select the ones exhibiting the highest HOTAIR and miR-204 expression for further study. The RT-qPCR results (Figure 3)

| Down-regulation of HOTAIR decreases the expression of HOXC8 and alters the expression of proteins related to cell proliferation, migration and invasion through up-regulation of miR-204
In order to ascertain as to whether HOTAIR or miR-204 could in-  group while the expression of miR-204 and the mRNA and protein expression levels of E-cadherin were notably elevated (all P < 0.05).
The aforementioned results provided evidence indicating that the down-regulation of HOTAIR decreases the expression of HOXC8 and alters the protein levels associated with cell proliferation, migration and invasion by up-regulating miR-204.

| Down-regulation of HOTAIR inhibits the proliferation of oesophageal cancer cells via upregulation of miR-204
The functions of HOTAIR and miR-204 on the viability of oesophageal cancer cells were examined through the application of a MTT assay. The MTT assay ( Figure 5A) results revealed there to be no significant difference in the EC9706 cells regarding the cell viability between the control group and NC group (P > 0.05), while reduced viability was identified in the si-HOTAIR group while enhanced levels were found in the HOTAIR group compared to the control group (both P < 0.05).
In the ECA109 cell line ( Figure 5B

| Down-regulation of HOTAIR suppresses migration and invasion of oesophageal cancer cells through up-regulation of miR-204
The effects of HOTAIR and miR-204 on migration and invasion of oesophageal cancer cells were subsequently investigated by scratch test and Transwell assay, the results of which are shown in Figure 6.
In the EC9706 cell line ( Figure 6A-D), no significant difference was identified regarding the scratch healing and invasion abilities of the cells between the control group and NC group (P > 0.05). However, in the si-HOTAIR group, the scratch healing and invasion abilities of the cells were inhibited when compared to the control group, while enhanced levels were observed in the HOTAIR group (all P < 0.05).
In the ECA109 cell line ( Figure 6E-H

| Down-regulation of HOTAIR blocks cell cycle progression and induces the apoptosis of oesophageal cancer cells through up-regulation of miR-204
Flow cytometry was employed in order to ascertain as to whether HOTAIR or miR-204 could affect the cell cycle distribution and apoptosis of oesophageal cancer cells. Cell cycle distribution after transfection revealed that there was no difference in cell cycle distribution between the control and NC groups (P > 0.05) in relation to the EC9706 cell line ( Figure 7A-D), In comparison to the control group, the proportion of cells at the G0/G1 phase remarkably increased in the si-HOTAIR group while the percentage of cells at the S phase had a significantly higher apoptosis rate (all P < 0.05); in the HOTAIR group, with fewer cells arrested at the G0/G1 phase but much more arrested at the S phase along with a reduced apoptosis rate (all P < 0.05).
In the ECA109 cell line ( Figure 7E-H

| Down-regulation of HOTAIR suppresses oesophageal cancer cell tumourigenicity through upregulation of miR-204
Finally, cell tumourigenicity was also assessed in order to elucidate the effects associated with HOTAIR or miR-204 on oesophageal tumourigenicity via xenograft tumour in nude mice. The results obtained in

| D ISCUSS I ON
Oesophageal cancer is a malignancy well-known for its aggressive nature. 27   In v a s io n a n d m ig r a ti o n P r o li fe r a ti o n A p o p to s is study asserted that patients with oesophageal squamous cell carcinoma exhibiting high HOXC8 expression levels had shorter median survival time when compared to those with poor levels of HOXC8 expression. 32 Another key finding of our study revealed that HOTAIR could bind to miR-204 and miR-204 could target HOXC8, while we identified that the down-regulation of HOTAIR resulted in a reduction in the expression of HOXC8 through the up-regulation of miR-204. These results suggested that HOTAIR could be a ceRNA of miR-204, which could inhibit HOXC8. Accumulated reports have demonstrated the role of HOTAIR in cellular processes as a ceRNA: HOTAIR can stimulate the development of glioma, serving as a ceRNA via sponging miR-126-5p 33 ; HOTAIR promotes to the progression of gastric cancer by acting as a ceRNA of miR-331-3p, which is mediating HER2. 34 Moreover, down-regulation of HOTAIR decreased the expression of Vimentin and MMP-9 but increased that of E-cadherin through up-regulation of miR-204. It has been revealed that HOTAIR could increase the expression of MMP-9 and hence promotes tumour aggressiveness. 35 Moreover, silencing of HOTAIR has been reported to aid in the up-regulation of E-cadherin while reducing the expression of Vimentin. 36 HOTAIR has been suggested to suppress the expression of E-cadherin in oral squamous cell carcinoma, thus stimulating tumour cell invasion and metastasis. 37 In addition, there were studies reporting that miR-204 could interact with lncRNA UCA1 and target HOXA10. 8,38 Another example revealed that metazoan miRs, via Our results further highlighted that the down-regulation of HOTAIR could inhibit proliferation, invasion and migration, while acting to suppress the apoptosis of oesophageal cancer cell lines and inhibit the tumour formation in nude mice through the up-regulation of miR-204. LncRNAs have been widely documented to play a crucial role in the regulation of basic biochemical and cellular activities. 43 Besides, lncRNA HOTAIR has been demonstrated to possess the ability to stimulate cancer metastasis by reprogramming the chromatin state. 44 Consistent with the observations of our study, a previous report indicated that the up-regulation of HOTAIR, with the aids of I-BET151 treatment, reduced the antiproliferative ability of the BET bromodomain inhibitor. 45 Additionally, from a breast cancer perspective, the overexpression of HOTAIR has been reported to promote cancer cell proliferation while the down-regulation of HOTAIR has been found to functionally reduce cancer cell growth as well as cell invasion in prostate cancer. 46,47 Apart from suppressing HOTAIR, up-regulation of miR-204 has been reported to inhibit tumour cell formation. A previous study revealed that miR-204, by targeting FOXM1, could act to suppress cell invasion in oesophageal cancer, 14 which was in consistency with the findings of our study. In addition, through the mediation of the SIRT1/p53 signalling pathway, miR-204 has been reported to promote emitochondrial apoptosis in doxorubicin-treated prostate cancer cells. 48 To conclude, this study illustrated that lncRNA HOTAIR could function as a ceRNA of miR-204, and the silencing of HOTAIR could reduce expression of HOXC8, which ultimately inhibited the proliferation, migration and invasion of oesophageal cancer cells (Figure 9). This suggested that repression of HOTAIR could be clinically helpful to suppress oesophageal cancer progression and HOTAIR could be a promising target for oesophageal cancer treatment. However, an additional molecular mechanism by which HOTAIR works has been reported involving mediation on epithelial genes expression (ie E-cadherin) through the recruitment of PRC2. 49,50 Therefore, more researches on mechanisms of HOTAIR-based oesophageal cancer therapeutics are required to explore the application potential.

ACK N OWLED G EM ENTS
We show sincere appreciation to the reviewers for critical comments on this article.

AUTH O R S CO NTR I B UTI O N S
AHW and PT designed the study. YZ and ZBY collated the data, carried out data analyses and produced the initial draft of the manuscript. XTZ and LNL contributed to drafting and polishing the manuscript. All authors have read and approved the final submitted manuscript.

CO M PE TI N G I NTER E S TS
The authors declare that they have no competing interests.