LncRNA-ANCR down-regulation suppresses invasion and migration of colorectal cancer cells by regulating EZH2 expression

Abstract

Our study aimed to explore the effects of long noncoding RNA (lncRNA)-ANCR on the invasion and migration of colorectal cancer (CRC) cells by regulating enhancer of zeste homolog 2 (EZH2) expression. CRC tissues and adjacent normal tissues were collected and CRC SW620 cells line and normal human intestinal epithelial cells (HIECs) were incubated. CRC SW620 cells line was transfected with ANCR-siRNA. The expressions of ANCR and EZH2 mRNA were measured by real-time quantitative polymerase chain reaction (RT-qPCR). EZH2 and trimethylation of H3K27 (H3K27me3) protein expressions were detected using Western blotting. The relationship between ANCR and EZH2 was determined through RNA pull-down and co-immunoprecipitation (co-IP) assays. Cell invasion and migration were determined by Trans-well and cell scratch assays. ANCR, EZH2 and H3K27me3 expressions were up-regulated in CRC tissues and SW620 cells (all $P<$ 0.05). After transfected with ANCR-siRNA, SW620 cells showed decreased ANCR expression and EZH2 mRNA and protein expressions (all $P<$ 0.05). According to the results of RNA pull-down and co-IP assays, ANCR could specifically bind to EZH2. The results of Trans-well and cell scratch tests showed that when ANCR expression was decreased, the invasion and migration abilities of SW620 cells significantly declined (both $P<$ 0.05). In conclusion, these results suggest that lncRNA-ANCR could influence the invasion and migration of CRC cells by specifically binding to EZH2.

1. Introduction

Colorectal cancer (CRC) is the third most common cancer in the world, accounting for more than 1 million new cases and 600,000 deaths every year [3]. It is said that about 50% of CRC patients develop metastases, and most of them have unresectable tumors [35]. Risk factors for CRC include high cholesterol, obesity, diabetes and atherosclerosis, which are components of a disease state called “metabolic syndrome” [26]. Incidence rates of CRC are the same in men and women partly because of historical changes in risk factors, such as increased use of aspirin, red meat consumption and decreased smoking, and improvements in treatment [38]. Most of the patients with metastatic CRC will receive chemotherapy, colonoscopic polypectomy or targeted biologic therapy (bevacizumab or an epidermal growth factor receptor monoclonal antibody) [27, 36, 42]. A study reveals that CRC is a kind of biologically heterogeneous disease that develops from the progressive accumulation of genetic changes, such as adenomatous polyposis coli (APC), PIK3CA, TP53, FBXW7 genes and long non-coding RNAs (lncRNAs) and epigenetic alterations, which leads to the transformation of normal colonic epithelium into colon adenocarcinoma [6, 39].

LncRNAs, a new class of functional RNAs, consist of more than 200 nucleotides and mainly transcribed by RNA polymerase II (Pol II) [43]. Many lncRNAs have been proven to be functionally associated with human diseases, especially cancers such as breast cancer, liver cancer, glioblastoma and leukaemia [10, 13]. ANCR is a member of lncRNAs which is found on human chromosome 4 upstream of USP46 gene and embeds MIR4449 and SNORNA26 within its 1 ${}^{\mathrm{st}}$ and 2 ${}^{\mathrm{nd}}$ introns [19]. It is said that ANCR can operate through distinct modes, including signals, molecular decoys, scaffolds for protein-protein interactions, and enhance RNAs to regulate diverse biological processes, including cell growth, transcriptional regulation, differentiation, invasion and metastasis [21, 31]. Enhancer of zeste homolog 2 (EZH2) is a kind of histone methyltransferase and possesses oncogenic properties; overexpressed EZH2 enhances cancer cell proliferation and invasion and promotes neoplastic transformation [17]. Previous studies have shown that ANCR could regulate osteoblast differentiation and promote bladder cancer metastasis by regulating EZH2 and runt-related transcription factor 2 (Runx 2) [2, 7]. To the best of our knowledge, there is no study covering the relationship between lncRNA-ANCR and EZH2 in regulating CRC progression. Therefore, in the present study, we hypothesized that ANCR expression was correlated with EZH2 and aimed to investigate the expressions of ANCR and EZH2 in CRC and the effects of lncRNA-ANCR on the invasion and migration of CRC cells through regulating EZH2 expression.

2. Materials and methods

2.1 Study subjects

A total of 122 CRC patients in the Harbin Medical University Cancer Hospital were collected during November, 2012 to November, 2015 for the study, including 68 males and 54 females, with an average age of (58.39 $\pm$ 13.47) years. The CRC specimens ( $n=$ 122) were collected and prepared from the patients. Adjacent normal tissues (10 cm beyond the cancer tissue) were collected during the same time. All the patients had complete clinical data, received no preoperative radiotherapy or chemotherapy and were pathologically confirmed with CRC, including invasive carcinoma ( $n=$ 12), ulcerative carcinoma ( $n=$ 76) and apophasis type carcinoma ( $n=$ 34). According to Dukes stage [12] and tumor nodes metastasis (TNM) staging system recommended by American Joint Committee on Cancer (AJCC) and Union for International Cancer Control (UICC) (2002) [1], 59 patients had lymphatic metastasis and 63 had no lymphatic metastasis; 18 patients were in stage I, 29 in stage II, 40 in stage III and 35 in stage IV. Histological grades were: well differentiated ( $n=$ 39), moderately differentiated ( $n=$ 50) and poorly differentiated ( $n=$ 33). This study was approved by the Ethics Committee of the Harbin Medical University Cancer Hospital and all the study subjects had signed informed consent.

2.2 Cell culture

The CRC cell lines including M5, HCT116, lovo, SW620, Caco-2, DLD1, HT29 and SW480 and normal human intestinal epithelial cells (HIECs) were purchased from the Cell Bank of Chinese Academy of Sciences (Shanghai, China). The cells were cultured overnight in Roswell Park Memorial Institute (RPMI) medium containing 10% fetal bovine serum (FBS) with penicillin (100 U/ml) at 37 ${}^{\circ }$ C in 5% CO ${}_2$ . After 48 h, the cells in the logarithmic growth phase were selected to extract RNA. Extended culture was continued on cell lines with high expression of ANCR measured by real-time quantitative polymerase chain reaction (RT-qPCR): CRC cell lines digested by trypsin-elhylene diamine tetraacetic acid (EDTA) (0.25%) and normal cells were transferred into large bottles (75 ml) for incubation. When filled up 70% of the bottle, the cells were digested by trypsin-EDTA (0.25%), counted, centrifuged for 5 min at 1000 rpm, and washed by RPMI medium for two times after the supernatant was discarded; after centrifuged for another 5 min at 1000 rpm, the cells were re-suspended in RPMI medium without serum and then cell suspension was made (1 $\times$ 10 ${}^8$ ml).

2.3 Cell transfection and grouping

The cells (1 $\times$ 10 ${}^4)$ were placed into a 24-well plate, and cultured overnight at 37 ${}^{\circ }$ C in 5% CO ${}_2$ for 12 h. siRNA and negative control vectors of ANCR were designed and prepared by Shanghai GenePharma Co., Ltd. (Shanghai, China). Cell transfection and grouping: (1) the cells were transfected with ANCR-siRNA (ANCR-siRNA group); (2) the cells were transfected with ANCR negative control (ANCR-NC group); (3) the cells received no transfection (blank group). After 48 h, the cells in the logarithmic growth phase were collected for further experiments. Transfection scheme of the ANCR-siRNA and ANCR-NC groups was as follows. The shRNA vector (1 $\mu$ g) was re-suspended in RPMI medium (50 $\mu$ l). Lipofectamine 2000 (Invitrogen Corporation, CA, USA) was diluted with 50 $\mu$ l RPMI medium, and 5 min later, Lipofectamine 2000 and RPMI medium were mixed and incubated at room temperature for 20 min. After the supernatant was removed, the cells were added with transfection complex mixture, and then incubated at 37 ${}^{\circ }$ C in 5% CO ${}_2$ for 6 h. Next, the transfection reagent was removed and RPMI medium was added for another incubation for 24 h. The cells were diluted (1:10) and transferred into another 24-well plate and incubated for 48 h. G418 (450 mg/L) was added for resistance selection. The cell transfection was observed under fluorescence microscope 4 to 6 days later. The positively cloned cells were diluted, added with G418 and incubated again for 48 h. The cells stably expressed in the logarithmic growth phrase were selected for further experiments.

2.4 Real-time quantitative polymerase chain reaction (RT-qPCR)

RNA extraction was carried out by RNeasy mini kit (Qiagen GmbH, Hilden, Germany). Reverse transcription of total RNA was conducted using TaqMan RNA Reverse Transcription (Applied Biosystems, Foster City, CA, USA). The reaction system was 15 $\mu$ l under the following conditions: at 16 ${}^{\circ }$ C for 30 min, at 42 ${}^{\circ }$ C for 30 min and at 85 ${}^{\circ }$ C for 5 min. RT-qPCR detection was performed using TaqMan Universal PCR kit (Applied Biosystems, Foster City, CA, USA). The reaction system (20 $\mu$ l) contained forward and reverse primers 2 $\mu$ l (5 $\mu$ M), Sybr green mix 12 $\mu$ l, template ( $\sim$ 10 ng) plus Rnase-free water 6 $\mu$ l under the following conditions: warm start for 10 min at 95 ${}^{\circ }$ C, 15 s at 95 ${}^{\circ }$ C, then 1 min at 60 ${}^{\circ }$ C, totally 40 cycles. RT-qPCR detection was carried out by ABI 7500 (Applied Biosystems, Foster City, CA, USA). Three holes were established for each sample, to determine ANCR and EZH2 expressions with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal reference gene. The primers used in this study were shown in Table 1. Relative quantitative analysis was determined by 2 ${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ .

2.5 Western blotting

The cells and tissues were washed with phosphate buffer saline (PBS), then added with cell lysate containing proper amount of protease inhibitor, shaken for 5 min at 4 ${}^{\circ }$ C and centrifuged (12000 $\times$ g) for 10 min at 4 ${}^{\circ }$ C. The supernatant was collected for protein extraction using Qproteome Mammalian Protein Prer kit (Qiagen GmbH, Hilden, Germany). After concentration was measured and balanced, the protein was added with 6 $\times$ buffer sample preparation, boiled and then stored at $-$ 20 ${}^{\circ }$ C for later use. A total of 50 $\mu$ g protein were used for sodium dodecyl sulfate polyacrylamide gel electropheresis (SDS-PAGE) and transferred to cellulose nitrate membrane (NCM). After blocked by milk, the membrane was incubated with mouse anti-human monoclonal antibody of EZH2 (1: 200; ab156269; Abcam Inc., Cambridge, MA, USA), and rabbit anti-human H3K27 trimethylation (H3K27me3) (1:1000; orb178007; Wuhan Booute Biotechnology Co., Ltd). The membrane was washed by Tris-Buffered Saline with Tween (TBST) for 4 times (each time for 10 min), then added with diluted (1: 200) goat anti-mouse IgG which was marked with IRDyeTM800DX for 1 h incubation at room temperature. The membrane was washed by TBST for another 4 times, followed by developing with developing substrate, using rabbit anti-mouse $\beta$ -actin monoclonal antibody as a reference. Quantification of protein bands was performed by LabWorks Image Acquisition and Analysis Software (UVP, Inc., Upland, CA, USA).

2.6 RNA-pull down and co-immunoprecipitation (co-IP)

RNA-pull down assay: Total cell RNA was extracted, followed by transcription for ANCR RNA fragment. Primers were independently designed for the two ends of the fragment and PCR was performed to amplify the fragment (The primer sequences were synthesized in vitro by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, China). In vitro transcription Kit Promega was used for in vitro transcription and biotin was used to label the fragment, followed by separation and purification. The purified biotin-labeled ANCR RNA fragment was incubated for 30 min at room temperature with cell protein extract (120 $\mu$ g of protein). Paramagnetic Streptavidin-Conjugated Dynabeads (Dynabeads; Dynal, Oslo, Norway) was used to absorb RNA protein complex.

Table 1

The primer sequences for real-time quantitative polymerase chain reaction (RT-qPCR)

Gene	Primer sequence
ANCR	F: 5’-GACATTTCCTGAGTCGTCTTCGAACGGAC-3’
	R: 5’-TAGTGCGATTTAGAGCTGTACAAGTTTC-3’
EZH2	F: 5’-TTGTTGGCGGAAGCGTGTAAAATC-3’
	R: 5’-TCCCTAGTCCCGCGCAATGAGC-3’
GAPDH	F: 5’-TGAACGGGAAGCTCACTGG-3’
	R: 5’-TCCACCACCCTGTTGCTGTA-3’

EZH2, enhancer of zeste homolog; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; F, forward; R, reverse.

Co-IP assay: Normal HIECs (3 $\mu$ g) was added with pre-cold co-IP cell lysate (Sigma-Aldrich Co. LLC., St. Louis, UAS) for dissociation at 4 ${}^{\circ }$ C for 1 h, followed by addition with protein A-Sepharose for prerinse, percussion on ice for 30 min and centrifugation (12,000 $\times$ g) for 30 min at 4 ${}^{\circ }$ C. Next, after the lysate was discarded, the HIECs were added with rabbit polyclonal antibody (2 $\mu$ g) and then shaken at 4 ${}^{\circ }$ C overnight. The next day, the HIECs were added and evenly mixed with protein A-Sepharose ball beads (50 $\mu$ l) at 4 ${}^{\circ }$ C for 2 h, followed by washing with lysate 4 times (each for 10 min) and addition with 2 $\times$ sample buffer for western blot assay.

2.7 Trans-well invasion assay

Trans-well chamber (Sigma-Aldrich Co. LLC., St. Louis, USA) was prepared. Upper chamber was glued with extracellular matrix (ECM) (50 $\mu$ l/hole) which was rested for 4 h at 37 ${}^{\circ }$ C. Liquid was sucked out and the residual liquid was left to be dried by air. Cells in ANCR-siRNA, ANCR-NC and blank groups were diluted to 1 $\times$ 10 ${}^5$ /ml, and incubated in culture medium without serum for 12 h. A total of 200 $\mu$ l cells were added into upper chamber and 600 $\mu$ l RPMI medium containing 10% FBS was added into the lower chamber. After 24 h, the cells in the lower chamber were taken out, fixed by ethanol (90%), dyed using crystal violet solution (0.1%) and then observed under microscope. Four low power fields (LPFs) ( $\times$ 100) were chosen for observation and counting and calculation for the average. The above experiments were repeated 3 times for each cell line.

2.8 Cell scratch test

The cell suspension (50 $\mu$ l) was taken out from each group (ANCR-siRNA, ANCR-NC and blank), placed into a culture dish (100 mm) and then added with RPMI medium without serum for incubation (12 h). A scratch was drawn with a 20 $\mu$ l suction head. The scratch was observed and photographed under microscope. The scratched cells were placed at 37 ${}^{\circ }$ C in 5% CO ${}_2$ for incubation for 72 h and observed every 24 h. The above experiments were repeated for 3 times for each cell line. The width of the scratch in each photo was analyzed.

2.9 Statistical analysis

Data was analyzed by SPSS 19.0 (SPSS Inc., Chicago, IL, USA). Enumeration data was expressed in rate or percentage, and comparison among groups was tested by chi-square test. Measurement data was expressed as mean $\pm$ standard deviation ( $\overline{x}\pm s$ ), and comparison between mean values of two samples was tested using $t$ -test. Multi-group comparison was analyzed through One-Way ANOVA (homogeneity of variance was tested before comparison) and pairwise comparison between mean values among multiple groups was analyzed by LSD-t test. A $P$ value of less than 0.05 was considered statistically significant.

Figure 1.

The protein expressions of EZH2 and H3K27me3 in CRC and adjacent normal tissues detected by Western blotting. CRC, colorectal cancer; EZH2, enhancer of zeste homolog 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Table 2

The expressions of lncRNA-ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein in CRC and adjacent normal tissues

	Adjacent normal tissues	CRC tissues	$P$ value
ANCR	0.11 $\pm$ 0.03	0.25 $\pm$ 0.12	0.037
EZH2 mRNA	0.43 $\pm$ 0.13	1.24 $\pm$ 0.46	0.016
EZH2 protein	0.36 $\pm$ 0.11	1.41 $\pm$ 0.52	0.003
H3K27me3 protein	0.41 $\pm$ 0.12	1.12 $\pm$ 0.21	0.019

CRC, colorectal cancer; EZH2, enhancer of zeste homolog.

Table 3

The correlations of the expressions of lncRNA-ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein with pathological features of CRC patients

Feature	n	ANCR	EZH2 mRNA	EZH2 protein	H3K27me3 protein
Age
$⩽$ 60	80	0.24 $\pm$ 0.12	1.22 $\pm$ 0.42	1.38 $\pm$ 0.48	1.09 $\pm$ 0.33
$>$ 60	42	0.27 $\pm$ 0.13	1.29 $\pm$ 0.53	1.47 $\pm$ 0.59	1.13 $\pm$ 0.18
Lymphatic metastasis
No	63	0.21 $\pm$ 0.11	1.16 $\pm$ 0.45	1.32 $\pm$ 0.51	0.97 $\pm$ 0.19
Yes	59	0.28 $\pm$ 0.13 ${}^{\ast }$	1.33 $\pm$ 0.46 ${}^{\ast }$	1.51 $\pm$ 0.51 ${}^{\ast }$	1.22 $\pm$ 0.28 ${}^{\ast }$
Dukes stage
Stage A-B	56	0.20 $\pm$ 0.10	1.15 $\pm$ 0.48	1.31 $\pm$ 0.54	1.02 $\pm$ 0.13
Stage C-D	66	0.29 $\pm$ 0.13 ${}^{\ast }$	1.32 $\pm$ 0.43 ${}^{\ast }$	1.50 $\pm$ 0.49 ${}^{\ast }$	1.20 $\pm$ 0.37 ${}^{\ast }$
TNM stage
I–II	47	0.19 $\pm$ 0.11	1.09 $\pm$ 0.41	1.31 $\pm$ 0.59	0.95 $\pm$ 0.36
III–IV	75	0.28 $\pm$ 0.12 ${}^{\ast }$	1.34 $\pm$ 0.46 ${}^{\ast }$	1.48 $\pm$ 0.46 ${}^{\ast }$	1.24 $\pm$ 0.40 ${}^{\ast }$
Histological grade
Low-differentiated	83	0.24 $\pm$ 0.12	1.23 $\pm$ 0.42	1.39 $\pm$ 0.48	1.07 $\pm$ 0.31
Well-differentiated	39	0.27 $\pm$ 0.14	1.28 $\pm$ 0.54	1.46 $\pm$ 0.61	1.15 $\pm$ 0.09
Pathological types
Invasive type	12	0.31 $\pm$ 0.15	1.62 $\pm$ 0.51	1.90 $\pm$ 0.86	1.03 $\pm$ 0.19
Ulcerative type	76	0.23 $\pm$ 0.11	1.18 $\pm$ 0.36	1.33 $\pm$ 0.32	1.09 $\pm$ 0.38
Apophasis type	34	0.26 $\pm$ 0.14	1.24 $\pm$ 0.58	1.42 $\pm$ 0.86	1.14 $\pm$ 0.15

CRC, colorectal cancer; EZH2, enhancer of zeste homolog; TNM, tumor node metastasis; ${}^{\ast }$ , $P<$ 0.05 compared with the patients with no lymphatic metastasis/Duke A-B/TNM I–II.

3. Results

3.1 The expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein in CRC and adjacent normal tissues

Table 4

The expressions of ANCR, EZH2 mRNA and EZH2 protein in CRC cell lines after transfection

Group	ANCR	EZH2 mRNA	EZH2 protein
ANCR-siRNA	0.56 $\pm$ 0.18 ${}^{\ast }$	0.68 $\pm$ 0.24 ${}^{\ast }$	0.71 $\pm$ 0.35 ${}^{\ast }$
ANCR-NC	1.14 $\pm$ 0.31	1.35 $\pm$ 0.52	1.52 $\pm$ 0.61
Blank	0.98 $\pm$ 0.25	1.26 $\pm$ 0.35	1.37 $\pm$ 0.54

CRC, colorectal cancer; EZH2, enhancer of zeste homolog 2; ${}^{\ast }$ , $P<$ 0.05 compared with the ANCR-NC and blank groups.

As shown in Table 2 and Fig. 1, compared with the adjacent normal tissues, the expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein were significantly higher in CRC tissues (all $P<$ 0.05). As shown in Table 3, the expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein were higher in patients with lymphatic metastasis than those without lymphatic metastasis (all $P<$ 0.05). According to Dukes stage, compared with A and B stages, patients in C and D stages had higher expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein (all $P<$ 0.05). Furthermore, patients with TNM stage III-IV CRC had higher expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein than those with TNM stage I-II CRC (all $P<$ 0.05). However, the expressions of ANCR, EZH2 mRNA, EZH2 protein and H3K27me3 protein showed no correlations with age, histological grade and pathological types of CRC patients (all $P>$ 0.05).

Figure 2.

The expressions of ANCR, EZH2 mRNA and EZH2 protein in eight CRC cell lines (M5, HCT116, lovo, SW620, Caco-2, DLD1, HT29 and SW480) and normal human intestinal epithelial cells (HIECs) after transfection. (A) The expressions of ANCR, EZH2 mRNA and EZH2 protein detected by RT-qPCR and Western blotting; (B) EZH2 protein expression detected by Western blotting; EZH2, enhancer of zeste homolog 2; HIEC, human intestinal epithelial cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ${}^{*}P<$ 0.05 compared with HIEC cells.

Figure 3.

The expression of EZH2 protein detected by Western blotting. EZH2, enhancer of zeste homolog 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

3.2 The expressions of ANCR, EZH2 mRNA and EZH2 protein in CRC cell lines after transfection

The results of RT-qPCR (Fig. 2) showed that ANCR and EZH2 mRNA expressions were higher in CRC cells (M5, HCT116, lovo, SW620, Caco-2, DLD1, HT29 and SW480) than those in normal HIECs (all $P<$ 0.05). Among eight CRC cell lines, SW620 cell line had the highest expressions of ANCR, EZH2 mRNA and EZH2 protein. Therefore, SW620 cell line was chosen to perform the next experiments. As shown in Table 4 and Fig. 3 ANCR-NC groups, SW620 cells in the ANCR-siRNA group exhibited significant decreases in the expressions of ANCR, EZH2 mRNA and EZH2 protein (all $P<$ 0.05). However, there was no difference in the expressions of ANCR, EZH2 mRNA and EZH2 protein between the blank and ANCR-NC groups (all $P>$ 0.05).

Figure 4.

The results of RNA pull-down and co-IP assays. (A) RNA expressions in ANCR and ANCR-control groups measured by RT-qPCR; (B) Western blotting for the expression of EZH2 protein; EZH2, enhancer of zeste homolog 2; ${}^{*}$ compared with ANCR-control, $P<$ 0.05.

3.3 The relationship between ANCR and EZH2

The results of RNA pull-down and co-IP assays indicated that ANCR could specifically bind to EZH2 (Fig. 4). The signal intensity of ANCR and EZH2 expressions were higher in the ANCR group than in the ANCR-control group (both $P<$ 0.05).

Figure 5.

Cell invasion ability of transfected cells in each group detected by Trans-well assay. (A) Cell invasion observed under the light microscope; (B) Cell counts in transfected cells. ${}^{*}P<$ 0.05 compared with the ANCR-NC and blank groups.

3.4 Effect of ANCR-siRNA on the invasion of SW620 cells

Trans-well invasion assay showed that ANCR expression was inhibited by siRNA, thus cell counts in the ANCR-siRNA group were lower than those in the ANCR-NC and blank groups (all $P<$ 0.05) (Fig. 5). Under the light microscopy, there were fewer cells, sparse and scattered in the ANCR-siRNA group. No differences were observed in cell counts and light microscopic morphology of SW620 cells between the ANCR-NC and blank groups ( $P>$ 0.05).

3.5 Effect of ANCR-siRNA on the migration of SW620 cells

As Fig. 6 and Table 5 showed, there was no evident difference in the scratch size among the three groups at 0 h and 24 h after transfection, but obvious differences were observed at 48 h and 72 h after transfection. Compared with the ANCR-NC and blank groups, migration distance of SW620 cells was significantly shorter in the ANCR-siRNA group (both $P<$ 0.05), which indicated that SW620 cells had higher migration ability in the ANCR-NC and blank groups than in the ANCR-siRNA group. There was no significant difference in migration distance of SW620 between the ANCR-NC and blank groups at 0 h, 24 h, 48 h and 72 h after transfection (all $P>$ 0.05).

4. Discussion

In this study we investigated the correlation between lncRNA-ANCR and EZH2 and explored how lncRNA-ANCR affected CRC cell invasion and migration through regulating EZH2. According to our study, ANCR expression was positively related to EZH2 expression and down-regulated ANCR decreased EZH2 expression, inhibiting invasion and migration abilities of CRC cells.

Table 5

The width of the scratch of SW620 cells in each group at 0 h, 24 h, 48 h and 72 h after transfection

Group	0 h	24 h	48 h	72 h
ANCR-siRNA	153.8 $\pm$ 21.5	148.7 $\pm$ 18.6	144.6 $\pm$ 16.4 ${}^{\ast }$	141.1 $\pm$ 17.7 ${}^{\ast }$
ANCR-NC	148.3 $\pm$ 13.4	139.4 $\pm$ 19.3	121.8 $\pm$ 16.6	101.2 $\pm$ 14.6
Blank	151.4 $\pm$ 16.8	141.9 $\pm$ 15.6	125.4 $\pm$ 13.9	98.6 $\pm$ 12.3

${}^{\ast }$ , $P<$ 0.05 compared with the ANCR-NC and blank groups.

Figure 6.

The migration of transfected cells in each group detected by scratch test.

We found that ANCR and EZH2 were highly expressed in CRC tissues, and correlated with lymphatic metastasis, Dukes stage and TNM stage. It was reported that ANCR was significantly up-regulated in cancer cells and/or tissues, such as gastric, breast and prostatic cancers as well as CRC [8, 13, 40]. Another study revealed that EZH2 was highly overexpressed in cancers, including CRC, and showed that EZH2 was integral to proliferation in cancer cells [31]. We have also determined H3K27me3 and found that it was highly expressed in CRC. H3K27me3 is a transcriptionally repressive epigenetic mark and has been causally involved in multiple solid and hematologic human cancers [21, 41]. Methylation of H3K27 is catalyzed by polycomb repressive complex 2 (PRC2), which contains the enzymatic subunit EZH2 [15, 30]. A previous study showed that high expression of EZH2 and H3K27me3 at the same time could serve as biomarkers in the prediction of esophageal squamous cell carcinoma (ESCC) metastasis and ESCC patients’ survival [22]. As our results showed, EZH2 and H3K27me3 were both found highly expressed in CRC.

According to the results, ANCR expression and EZH2 mRNA and protein expressions were down-regulated in the SW620 cells transfected with ANCR-siRNA, indicating that when ANCR was inhibited, EZH2 expression was down-regulated; therefore, we assumed that ANCR could specifically bind to EZH2. The result echoed a previous study which showed that there was a higher enrichment of ANCR with the EZH2 antibody than with non-specific IgG antibody and a 305-nt region at the 30 end of ANCR was required for its association with EZH2, indicating that ANCR was specifically associated with EZH2 [44]. The mechanism of how lncRNA-ANCR regulated EZH2 expression may be associated with PRC2. LncRNAs have been popularly reported to interact with PRC2 and facilitate its recruitment to promoter of some target genes [24]. LncRNAs can be functionally classified into structural, repressive and activating ones. Repressive lncRNAs mediate their actions in many ways, such as recruiting repressive complexes at the target loci and causing transcriptional interference, to subsequently suppress transcription or prevent the formation of transcription initiation complex at the target loci [20]. Braveheart lncRNA, which is demonstrated to activate gene expression, is recently reported to interact with SUZ12 (a member of PRC2), which is supposed to prevent PRC2 from acting on the target loci [18]. EZH2 is the catalytic component of PRC2, functioning as a H3K27 methyltransferase; H3K27me3 correlates with transcriptionally repressed chromatin [16].

In our study, the invading cells were found sparse and scattered in SW620 cells transfected with ANCR-siRNA, and the migration was also inhibited, indicating that down-regulated ANCR could suppress the invasion and migration abilities of CRC SW620 cells. Consistently, Liu et al. reported that high expression of lncRNA-DANCR was involved in the progression of hepatocellular carcinoma [23]; Prensner et al. demonstrated that lncRNA-PCAT-1 was overexpressed in high-grade and metastatic tumors [28]; and it was showed that knockdown of lncRNA-HOTAIR could inhibit cell invasion in breast cancer [9]. Furthermore, according to the result in our study, ANCR regulated CRC cell invasion and migration through regulating EZH2. Consistently, a study showed that ANCR could reduce osteogenic differentiation through a mechanism suggested to involve EZH2 [34]. High expression of EZH2 is reported to be associated with the invasion and migration in many cancers, including hepatocellular carcinoma, prostate cancer, endometrial cancer and nasopharyngeal carcinoma [4, 11, 25, 29]. E-cadherin (E-cad), a cell-to-cell adhesion molecule, is associated with the invasion and metastasis of tumor cell [33]. Compelling evidence has showed that E-cad expression is repressed in cancer, suggesting its potentially critical role in the malignant progression of epithelial tumors [5]. EZH2 has been reported to suppress E-cad expression, which promotes metastasis of oral tongue squamous cell carcinoma [37]. Cao et al. demonstrated that EZH2 could mediate transcriptional silencing of the tumor suppressor gene E-cad by H3K27me3 [5]. Based on all the statement here, we may have not much difficulty to understand the suppression of the invasion and migration abilities of CRC SW620 cells in the ANCR-siRNA group. In the ANCR-siRNA group, ANCR was up-regulated as well as EZH2. Up-regulated EZH2 may inhibit E-cad expression and therefore the invasion and migration were suppressed.

Taken as a whole, our study revealed that ANCR and EZH2 have high expressions in CRC. Furthermore, lncRNA-ANCR could influence the invasion and migration of CRC cells by specifically binding to EZH2, which provided a reference for treatment of CRC. Our study had several limitations: firstly, we just investigated the correlation between lncRNA-ANCR, EZH2 and CRC; the mechanism of how decreased lncRNA-ANCR-meditated EZH2 declined CRC cell invasion and migration needed to be further studied. The small sample size of the present study was also a limitation which might likely lead to a not very convincing conclusion. All of these help to provide great chance for us to improve our researches in the future.