HDAC2 promotes the EMT of colorectal cancer cells and via the modular scaffold function of ENSG00000274093.1

Histone deacetylase 2 (HDAC2), a member of the Histone deacetylase family, plays a vital role in various carcinomas. In this study, we identified that HDAC2 expression levels are associated with liver metastasis, higher T stages and poor prognosis in colorectal cancer. HDAC2 down‐regulation via lentivirus‐mediated expression of HDAC2‐targeting shRNA reduced the in vitro migration and invasion ability of HCT116 cell as well as their liver metastasis in nude mouse xenografts. Mechanistically, HDAC2 promotes epithelial‐mesenchymal transition (EMT) in colorectal cancer cells by combining HDAC1 with EZH2 (a key histone methyltransferase), possibly through the modular scaffold function of a new lncRNA, ENSG00000274093.1. HDAC2 thus appears to promote CRC cell migration and invasion through binding HDAC1 and EZH2 via ENSG00000274093.1.


| INTRODUC TI ON
Colorectal cancer (CRC) is the third most commonly diagnosed cancer, with an estimated 1.1 million cases and 551 269 deaths in 2018, 1 and liver metastasis is the main cause of death in patients with colorectal cancer. 2 Therefore, to improve the diagnosis and treatment of colorectal cancer, researching the liver metastasis of CRC is necessary. Recent studies revealed histone acetylation, one of the most extensively studied histone modifications, is regulated by the histone deacetylases (HDACs), and play a pivotal role in cancer development and progression. 3,4 Given the fact that the activity of HDACs is dysregulated in many types of cancer, thus HDACs have been considered as therapeutic targets for the treatment of cancers.
HDAC2 has reportedly overexpressed in liver, cervical and lung cancers. [5][6][7] There is also a high expression of HDAC2 in the carcinogenesis of colorectal cancer. Anne et al analysed 254 cases of colorectal cancer and 50 cases of normal colorectal mucosa. The results showed that there was an increase of HDAC2 and an abnormal level of H3K56 acetylation in colorectal cancer tissues. 8 Zhu and other studies have shown that HDAC2 plays an important role in the development of colorectal cancer and can be used as a potential therapeutic target. 9 However, at present, there is still a lack of research on the role of HDAC2 in colorectal liver metastasis and its regulatory mechanism.
LncRNA is a class of non-coding transcripts greater than 200 nucleotides in length. Recent studies have revealed that lncRNAs may affect development, metabolism and other aspects of cancer. 10,11 The role of lncRNAs and the underlying mechanisms in CRC also has been reported before. [12][13][14][15] However, more specific mechanisms of lncRNAs in CRC need to be further teased out.
In the present study, we found that HDAC2 was highly expressed in colorectal cancer compared to adjacent normal mucosa.
Clinicopathologic data indicated that higher HDAC2 expression correlated with poor overall survival (OS) and was associated with liver metastasis and higher T stages. Besides, HDAC2 knockdown prevented cell migration and invasion. This process requires a novel LncRNA, ENSG00000274093.1, which binds to HDAC2 and may act as a modular scaffold for the HDAC1/HDAC2 and EZH2 complexes, thereby altering EMT in colorectal cancer cells.

| Patient specimens
Tumours and the adjacent colorectal tissues were obtained from patients with colorectal cancer who underwent surgery at Zhongshan Hospital, Fudan University, from June 2016 to July 2016. The study protocol was approved by the ethics committee of Zhongshan Hospital, Fudan University. Written informed consent was obtained from all participants in this study. All the research was carried out in accordance with the provisions of the Declaration of Helsinki. None of these patients had received radiotherapy or chemotherapy before surgery. The percentage of tumour cellularity in the tissue section was at least 70% as determined by a pathologist.

| Immunohistochemistry
HDAC2 expression was measured in tumour samples using a Histostain®-Plus 3rd Gen IHC Detection Kit (Invitrogen, USA #85-9073) following the manufacturer's instructions. The tissue microarray (TMA) slide was dried overnight at 37°C, deparaffinized in xylene, rehydrated through graded alcohol, immersed in 3% hydrogen peroxide for 20 minutes to block endogenous peroxidase activity and then antigen-retrieved by microwave heating in 0.01 M sodium citrate buffer (pH = 6.0) at 100°C for 30 minutes. Subsequently, slides were pre-incubated with 10% normal goat serum at room temperature (RT) for 30 minutes to reduce the non-specific reaction. The primary rabbit anti-HDAC2 polyclonal antibody (ab41587, Abcam) was diluted (1:50) in phosphate buffered saline (PBS) with 3% bovine serum albumin (BSA) and applied overnight in a humidity chamber at 4°C. Slides were incubated with a polymer peroxidase-labelled secondary antibody for 30 minutes at RT and then stained with DAB. Finally, sections were counterstained with haematoxylin.
The expression of HDAC2 was determined by the positive tumour cells proportion and the average intensity of positive tumour cells in the tumour tissues. Four levels were defined: no staining = -, weak staining = +, moderate staining = ++ and strong staining = +++. The final score was classified as low or high expression using the median value as the cut-off.

| Cell culture
The human colorectal cancer cell lines HCT116 and LoVo were obtained was maintained in DMEM medium supplemented with 10% foetal bovine serum and 100 units/ml penicillin-streptomycin (Invitrogen, USA) at 37°C in 5% CO 2 .

| Co-immunoprecipitation
Co-immunoprecipitation was performed as described previously. In brief, both input and IP samples were analysed by Western blot using various antibodies at the indicated dilutions: anti-HDAC1 antibody (1:1,000; Cell Signal Technology), anti-EZH2 antibody (1:1,000; Cell Signal Technology) and normal rabbit IgG.

| RNA immunoprecipitation
RNA immunoprecipitation (RIP) experiments were performed using the Megna RIP RNA-binding Protein Immunoprecipitation Kit (Millipore). The anti-HDAC2 antibody used for RIP was purchased from CST (Cell Signaling Technology). The co-precipitated RNAs were detected by reverse transcription PCR. To demonstrate that the detected RNA signals specifically bind to HDAC2, total RNA (input controls) and normal rabbit IgG controls were simultaneously assayed.

RNA Analysis, Extraction and Quantitative Real-Time PCR:The
RNA expression levels were measured using a real-time quantitative PCR system. Total RNA was extracted by TRIzol reagent (Invitrogen), and 1 μg of total RNA was reverse-transcribed using the PrimeScriptP RT Reagent Kit (Perfect Real Time; Takara). The amplified transcript level of each specific gene was normalized to that of 18S of GAPDH.
The primers were provided by Shenggong Company (Table S1).

| RNA pull-down assay
Cell nuclear lysates were incubated with biotinylated RNA and streptavidin beads for RNA pull-down incubation. Then, beads were collected by centrifugation. RNA-associated proteins were eluted and resolved by SDS-PAGE followed by silver staining (Bio-Rad).

| Fluorescence in situ hybridization (FISH) assay
Cells grown on coverslips were fixed with 4% paraformaldehyde at 25°C for 15 minutes, washed three times with PBS and treated with 0.5% Triton X-100 at room temperature for 10 minutes. The samples were dehydrated in 3% H 2 O 2 and air-dried. After probe hybridization solution was added, the samples were mounted, denatured at 88°C for 5 minutes, and hybridized in a humid and dark environment at 37°C
Cells at logarithmic phase were transfected with lentivirus-mediated knockdown of HDAC2, and knockdown or overexpression of ENSG00000274093.1 or control HCT116 (Genechem, Shanghai).

| Statistical analysis
Comparisons between groups were performed using paired Student's t tests, and differences were considered statistically significant at P < 0.05. Data were presented as the mean ± SD of three independent experiments.

| HDAC2 was highly expressed in colorectal cancer tumour tissues and was associated with poor overall survival
HDAC2 protein expression in the primary tumour tissue samples from 121 patients was detected by immunohistochemistry (IHC).
Representative images ( Figure 1A) show four different typical F I G U R E 1 HDAC2 was highly expressed in colorectal cancer tumour tissues and was associated with poor overall survival. (A) Representative images of HDAC2 IHC staining intensity: (−) no staining; (+) weak staining; (++) moderate staining; (+++) strong staining. (B) HDAC2 IHC staining score was significantly higher in tumour tissues than in normal tissues. **<0.01; ***<0.001. (C) Kaplan-Meier survival curve with HDAC2 expression in tumour tissues for CRC patients after primary tumour resection. HR: hazard ratio; CI: confidence interval; P value: log-rank test HDAC2 expression patterns. HDAC2 IHC scores in tumours with liver metastasis were significantly higher than those in tumours without liver metastasis (P < 0.01) and normal tissues (P < 0.001) ( Figure 1B), suggesting that HDAC2 accumulated in CRC patients, especially in ones with liver metastasis.
For the IHC score, the median value was defined as the cut-off between the HDAC2 high-and low-expression subgroups. A total of 70 samples exhibited high expression, and 51 exhibited low expression. High HDAC2 expression was associated with primary pT stage 3/4 (P = .023) and liver metastasis (P < 0.001) ( Table 1).
The median follow-up time for all patients was 51.0 months and 58.3%, respectively. For patients with low HDAC2 expression, they were 87.3% and 74.6%, respectively ( Figure 1C). These results suggested that higher HDAC2 expression was associated with poorer OS. *P < 0.05, **P<0.01, statistically significant.

| HDAC2 down-regulation inhibited tumour cell migration and invasion via EMT in vitro andin vivo
HCT116 cells were infected with lentivirus containing either shRNA targeting HDAC2 or control scrambled shRNA. Western blotting and real-time PCR analysis showed that HDAC2 effectively decreased both HDAC2 protein and mRNA levels ( Figure S1). In transwell-migration model, the results of 10 × microscopic observation showed that the number of transmembrane of HCT116 cells decreased significantly after HDAC2 down-regulation (148.3 vs. 113.7, P = .0347).
In the transwell-invasion model, it gets the same result (88.7 vs. 54.7, P = .0229) (Figure 2A-B) . All wound healing images showed that HDAC2 down-regulation suppressed the cell migration ability and this effect was obviously concentration-and time-dependent ( Figure 2C). To address whether HDAC2 modulates tumour cell migration and invasion, the levels of the EMT marker, Vimentin and E-cadherin, were detected. When HDAC2 down-regulation, the expression of Vimentin was up-regulated, whereas the expression of E-cadherin was down-regulated ( Figure 2D).
HCT116 cells stably expressing control or HDAC2 shRNA were injected into the splenic capsule of nude mice to form xenograft tumours.

In addition, the expression of Vimentin was up-regulated, whereas the expression of E-cadherin was down-regulated in tumours infected with
Lv-shHDAC2 as detected by IHC ( Figure 2F). Taken together, these results suggest that HDAC2 may be a tumour metastasis-promoting oncogene, and this process was closely connected with EMT.

| ENSG00000274093.1 was binding to HDAC2 and was highly expressed in colorectal cancer tumour tissues
For the mechanism of HDAC2 affecting EMT, Burstin et al found that HDAC2 bind to HDAC1 and then bind to Snail to up-regulate the expression of E-cadherin, thus promoting the occurrence of EMT in tumour cells. 16 Subsequently, Tong et al found that EZH2 was also indispensable in the process of HDAC2/HDAC1/Snail complex affecting the expression of E-cadherin. And HDAC2/HDAC1 complex plays a bridge role in the binding process of EZH2 and Snail. 17 Moreover, small molecules could play a significant role in the process of the function of HDACs. 18-20 Therefore, we evaluated whether RNA was needful for HDAC2/HDAC1 and EZH2 binding. Co-Immunoprecipitation experiments showed that the binding of HDAC2/ HDAC1 and EZH2 decreased after RNase treatment in HCT116 cells ( Figure 3A).
To determine the crucial RNA, especially LncRNA, an HDAC2-RIP-seq experiment was conducted. After analysis, the top 20 enrichment lncRNAs were selected ( Figure 3B) increased, which may be consistent to the progress of colorectal cancer ( Figure 3C & Figure S2). And lncRNAs pull-down showed that HDAC1 and EZH2 could bind to ENSG00000274093.1, rather than ENSG00000280408.1, ENSG00000259715.1 ( Figure 3D).
LncRNA ENSG00000274093.1, located on chr16:69632141-69632571:1, is a kind of sense intronic lncRNA. The ENSG00000274093.1 has its copy from the introns of NFAT5 that does not overlap any exons.( Figure S3) The distribution of lncRNAs in cells is different, and the function mechanism is also different. 11 We found that ENSG00000274093.1 was mainly located in the cell nucleus ( Figure 3E). This suggests that ENSG00000274093.1 may play a role in regulating the binding of HDAC2, HDAC1 and EZH2.  Figure 4E).