Long non‐coding RNA HEIH suppresses the expression of TP53 through enhancer of zeste homolog 2 in oesophageal squamous cell carcinoma

Abstract It is increasingly evident that the molecular and biological functions of long non‐coding RNAs (lncRNA) are vital for understanding the molecular biology and progression of cancer. The lncRNA‐HEIH, a newly identified lncRNA, has been demonstrated to be up‐regulated in hepatocellular cancer. However, little is known about its role in oesophageal squamous cell carcinoma (ESCC). In the present study, an obvious up‐regulation of lncRNA‐HEIH was observed in ESCC compared to the adjacent normal tissues. Meanwhile, patients with high expression of lncRNA‐HEIH have significantly poorer prognosis than those with low expression. We further found that lncRNA‐HEIH was associated with enhancer of zeste homolog 2 (EZH2) and that this association led to the repression of TP53. These findings indicate that lncRNA‐HEIH may serve as a prognostic marker and a potential therapeutic target for ESCC.


Funding information
This work was supported by grants from the Shanghai technological foundation (12JC1410900) and the National Natural Science Foundation of China (81402449).

Abstract
It is increasingly evident that the molecular and biological functions of long non-coding RNAs (lncRNA) are vital for understanding the molecular biology and progression of cancer. The lncRNA-HEIH, a newly identified lncRNA, has been demonstrated to be up-regulated in hepatocellular cancer. However, little is known about its role in oesophageal squamous cell carcinoma (ESCC). In the present study, an obvious upregulation of lncRNA-HEIH was observed in ESCC compared to the adjacent normal tissues. Meanwhile, patients with high expression of lncRNA-HEIH have significantly poorer prognosis than those with low expression. We further found that lncRNA-HEIH was associated with enhancer of zeste homolog 2 (EZH2) and that this association led to the repression of TP53. These findings indicate that lncRNA-HEIH may serve as a prognostic marker and a potential therapeutic target for ESCC.

K E Y W O R D S
lncRNA-HEIH, long non-coding RNA, oesophageal squamous cell carcinoma, TP53, zeste homolog 2 In our study, we investigated whether expression of the HEIH gene was associated with ESCC growth and metastasis.
Accordingly, the level of HEIH in ESCC tissues was detected and its potential relationship with clinical pathologic parameters and tumour recurrence was analysed. The role of lncRNA-HEIH in growth and metastasis during progression of ESCC was also studied both in vitro and in vivo.

| Real-time PCR (RT-PCR) and Western blot analysis
We employed RT-PCR analyzation to measure the expression levels of RNAs. Total RNA was isolated using TRIzol (Invitrogen), and genomic DNA was removed by RNase-free DNase. First-strand cDNA was generated with the Prime Script RT reagent kit (Takara, Dalian, PR China).
Step One™ Real-Time PCR System (Applied Biosystems, Foster City, USA) and gene-specific primers were applied for RT-PCR. Gene expression in each sample was normalized to 18S rRNA expression. Significance was determined by taking the average of the 18S rRNA-normalized 2 -ΔΔCT values. The gene-specific primers can be seen details in Table S1.

| Cell proliferation assay
A total of approximately 3.0 × 10 3 ESCC cells were plated in 96-well plates. Cell proliferation was assessed using the Cell Counting Kit-8 (Beyotime, Jiangsu, China) according to the manufacturer's protocol.
All of the experiments were performed in triplicate. The cell proliferation curves were plotted based on the absorbance at each time point.

| Small interfering RNA (siRNA)
To inhibit EZH2, 50 nmol/L EZH2 siRNA (Santa cruze) was trans-   The co-precipitated RNAs were detected by reverse transcription PCR. Total RNAs (input controls)and isotype controls were assayed simultaneously to demonstrate that the detected signals were from RNAs specifically binding to EZH2 (n = 3 for each experiment). The gene-specific primers used for detecting lncRNA-HEIH could refer to Table S2.

| Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed with the EZ ChIP™ Chromatin Immunoprecipitation Kit (Millipore Bedford, MA, USA) in line with its manual. Briefly, cross-linked chromatin was sonicated into fragments with 200-1000 bp. The chromatin was immunoprecipitated by anti-Ezh2 (clone AC22) and anti-H3K27me3 (Millipore). Normal mouse IgG was applied as a negative control.
Quantitative PCR was conducted using SYBR Green Mix (Takara Bio, Otsu, Japan). Primer sequences are listed in Table S2.

| Microarray analysis
Gene expression profiles of the Eca109 cells with or without lncRNA-HEIH overexpression were determined by Phalanx human One Array microarrays (HOA 6.1) following the manufacturer's instructions.
Gene-gene interaction network was constructed based on the data of differentially expressed genes. Network maps were constructed via Java which allows users to build and analyse molecular networks. For instance, if there is confirmative evidence that two genes interact with each other, an interaction edge is assigned between them. The considered evidence is from the interaction database from KEGG. Networks are stored and presented as graphs, where nodes are mainly genes (protein, compound, etc) and edges represent relation types between the nodes, for example activation or phosphorylation. The graph nature of networks raised our interest to view the networks as a powerful tool implemented in R.
To investigate the global network, we computationally identify the critical nodes. To this end, we defined the connectivity (also known as degree) as the sum of connection strengths with the other network genes: In gene networks, the connectivity measures how correlated a gene is with all other network genes. For a gene in the network, the number of source genes of a gene is called the indegree of the gene and the number of target genes of a gene is its outdegree. The character of genes is described by betweenness centrality measures reflecting the importance of a node in a graph relative to other nodes.
For a graph G:(V, E) with n vertices, the relative betweenness cen-

| Statistical evaluation
All the statistical analyses were conducted by SPSS version 17.0 software. For comparisons, one-way analyses of variance, Fisher's exact tests, chi-squared tests and two-tailed Student's t tests were performed as appropriate. The Kaplan-Meier method was included to evaluate the cumulative survival probability was evaluated using the Kaplan-Meier method, and differences were assessed using the log-rank test.

| LncRNA-HEIH expression is up-regulated in human ESCC tissues
Quick RT-PCR analysis was used to measure lncRNA-HEIH level in 91 ESCC tissues and normal counterparts. The expression of lncRNA-HEIH was significantly up-regulated in ESCC tissues ( Figure 1A). Furthermore, correlation analysis of lncRNA-HEIH expression with clinical pathological features of ESCC patients revealed a significant association between lncRNA-HEIH up-regulation and advanced pathological stage, tumour invasion and lymph node metastasis (Table 1). However, lncRNA-HEIH expression was not correlated with histological subtype and gender. Kaplan-Meier survival analysis and log-rank tests on patients postoperative survival were performed to further evaluate the correlation between lncRNA-HEIH expression and ESCC patient prognosis. According to the median ratio of relative lncRNA-HEIH expression in tumour tissues, the 91 ESCC patients were classified into two groups: High-lncRNA-HEIH group (n = 46, lncRNA-HEIH expression ratio ≥ mean ratio) and Low-lncRNA-HEIH group (n = 45, lncRNA-HEIH expression ratio < mean ratio). The Kaplan-Meier survival curve showed that patients with high-lncRNA-HEIH expression levels evidently had shorter tumour-free and overall survival time than those with low levels ( Figure 1B and C). These findings support the hypothesis that high level of lncRNA-HEIH expression plays a key role in ESCC progression.

| LncRNA-HEIH promotes the cell proliferation in ESCC cells
To evaluate the influence of lncRNA-HEIH on the biological behaviours of oesophageal cancer cells, we constructed cell lines with lncRNA-HEIH stable overexpression and down-expression ( Figure S1). Cell counting kit-8 assays indicated that the proliferation of oesophageal cancer cells was increased in Eca-109 cells and TE13 cells when lncRNA-HEIH was overexpressed and reduced in both cells when lncRNA-HEIH was knocked down (Figure 2A,B).

| A wide range of gene expressions was altered after lncRNA-HEIH overexpression
DNA microarray data showed that lncRNA-HEIH overexpression induced widespread changes in gene expression profile of cell line Eca-109, with 449 genes up-regulated and 419 down-regulated ( Figure 3A). The SAS system was used for gene ontology analysis (GO analysis) of the differentially expressed genes and P ≤ .05 was considered to be statistically significant (Table S3) (Table S4).
Additionally, to determine the regulatory relationships of these differentially expressed genes and the key players in ln-cRNA-HEIH-related pathways, we performed a network analysis to generate an interaction network containing relevant biological information for the 449 up-regulated and 419 down-regulated genes. The resulting network shows a high degree of connectivity that further supported the existence of biologically related functions ( Figure 3B).
According to the connected subgraphs and their GO terms, the functional modules were enriched for the p53 signalling pathway.
Some critical genes are located in these modules, including TP53 ( Figure 3B), suggesting that TP53 may have important roles in the lncRNA-HEIH regulated signalling pathways.
TP53 expression between lncRNA-HEIH overexpression Eca-109 and control cells was validated by RT-PCR ( Figure 3C). We found that there were significantly lower levels of TP53 in lncRNA-HEIH overexpression Eca-109 cells. We further performed Western blot analysis on TP53 ( Figure 3D) and drew a conclusion that there was a good correlation between the protein level and the gene expression data.

| LncRNA-HEIH inhibiting TP53 expression in ESCC tissues
We also investigated whether the mRNA expression of TP53 was inversely correlated with the levels of lncRNA-HEIH in ESCC tissues.

| D ISCUSS I ON
LncRNAs have been estimated to regulate many human genes and control a variety of cellular processes. 14 Recent studies have also shown that lncRNAs are deregulated in various cancers and their expression is relevant to the diagnosis and prognosis of a diverse array of tumours. 15,16 Even though various publications have studied the function of lncRNAs, the relation between lncRNA-HEIH and ESCC remains unknown. In our study, we first demonstrated that lncRNA-HEIH is significantly up-regulated in human ESCC tissues. We also found that altered lncRNA-HEIH expression levels are associated with the ESCC tumour invasion and clinical stage. We Recently, many studies showed the function of lncRNAs as drivers of tumour oncogenic and play suppressive roles in ESCC. [19][20][21] Previous reports have suggested that lncRNA-HEIH was up-regulated in hepatocellular carcinoma and associated with PRC2 to epigenetically regulate cell cycle-related genes. 13 In our study, we

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. Project administration (equal); Resources (equal); Validation (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data are free access to available upon request. were conducted on the lncRNA-HEIH promoter regions using anti-EZH2 and H3K27me3. Enrichment was determined relative to input controls. The data are the means ± standard deviations of three independent experiments