Five CRC cell lines (HT-29, SW480, HCT116, CaCo-2, and LoVo) were used. The CRC cell lines, HT-29 and HCT116, were provided by Professor Q. Tao at the Chinese University of Hong Kong, and the SW480, CaCo-2, and LoVo cell lines were purchased from the Chinese Academy of Sciences. The cell lines were cultured with RPMI-1640 medium (Gibco-BRL, Karlsruhe, Germany) containing 10% fetal bovine serum (FBS) (ExCell Bio, Shanghai, China), and cultured in a 5% CO₂ incubator at 37°C. The CRC tissues and paracarcinoma tissues, which were diagnosed by a pathologist, were obtained from patients during surgery at the First Affiliated Hospital of Chongqing Medical University (Chongqing, China). All patients signed an informed consent form, and the research protocol was approved by the Institutional Ethics Committee of the First Affiliated Hospital of Chongqing Medical University.

Total RNA was extracted separately from 32 paired CRC tissues, surgical margin tissues, and five CRC cell lines using TRIzol^® reagent (Life Technologies, Carlsbad, CA, USA). Genomic DNA was separately obtained from 24 CRC tissues, six normal colorectal tissues, and the five CRC cell lines using the QIAamp DNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. The concentration of DNA and RNA were measured using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Rockford, IL, USA) and stored at −80°C. The experimental and control group of the HT-29 and SW480 cells were lysed using a protein extraction reagent (Thermo Scientific) that contained the protease inhibitor, phenylmethane sulfonyl fluoride, and a phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and the lysate was then homogenized using a Ultrasonic Cell Grinder (Scientz, Ningbo). The supernatant was collected after centrifugation, and the concentration of protein in the supernatant was determined using the BCA protein kit (Thermo Scientific).

ZNF545 expression in CRC cells and tissues was determined using semiquantitative PCR and quantitative PCR. The RNA (1 µg) was reverse transcribed to 20 µg of cDNA using the Reverse Transcription system (Promega, Madison, WI, USA). For semiquantitative PCR, the ZNF545 gene was amplified using GoTaq DNA polymerase (Promega) with 35 cycles, using GAPDH as an internal control. The primer sequences are listed in Table I. Quantitative PCR was performed with a SYBR mix green fluorescent reagent (Promega) using an ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA, USA). β-actin was used as a control, and each sample was tested in triplicate.

As previously described (20), methylation-specific PCR (MSP) was used to detect the ZNF545 promoter methylation status. bisulfite-treated DNA (50 ng) was mixed with AmpliTaq Gold polymerase (Applied Biosystems), mgCl₂, and deoxynucleotide triphosphates for the MSP amplification reaction. The methylation-specific primers are listed in Table I. The PCR amplification was performed for a total of 40 cycles with an annealing temperature of 60°C or 58°C for 30 sec. The final products were analyzed on a 2% agarose gel, and then recorded using a Molecular Imager (Bio-Rad, Hercules, CA, USA).

Aza, a DNA methyltransferase (DNMT) inhibitor, makes DNMT inactivation through DNMT covalent bonding with thiol on cysteine residues, causing reactivation genes silenced by promoter methylation. TSA, a histone deacetylase inhibitor, plays an important role in controlling the tightness of DNA around histone. Combination treatment of Aza and TSA leads the synergistic activation of methylated genes.

CRC cell lines, SW480 and HT-29, were cultured and demethylated. The cells were treated with Aza (Sigma-Aldrich) in the dark at a final concentration of 10 mM/l for 3 days or TSA at a final concentration of 100 mM/l for 1 day, and further treated with or without 100 mM/l TSA (Cayman Chemical, Ann Arbor, MI, USA) for another 1 day.

A ZNF545-expressing plasmid was provided by Professor Q. Tao at the Chinese University of Hong Kong. After SW480 and HT-29 cells were plated into 6-well plates, the cells were transfected with pcDNA3.1-ZNF545 or pcDNA3.1 (+) vectors using Lipofectamine^® 2000 reagent (Invitrogen) and cultured with serum-free RPMI-1640 medium. After 4–6 h, the medium containing 10% FBS was changed to a selection medium containing 400 µg/ml G418 (Invitrogen/Gibco) and incubated for 48 h. Approximately 14 days later, the HT-29 and SW480 cells were stably expressing pcDNA3.1 and pcDNA3.1-ZNF545.

SW480 and HT-29 cells stably expressing ZNF545 or pcDNA 3.1 were seeded into 96-well plates at 2,000 cells/well, and the cell viability was measured using MTS (Promega) at 0, 24, 48 and 72 h using a Multiskan Spectrum™ (Tecan, Switzerland). The stably expressing cells were counted and seeded into 6-cell plates at 500 cells/well. Visible cell clones appeared in ~2 weeks, then they were fixed with 4% paraformaldehyde for 30 min and then stained with crystal violet solution for 30 min. Finally, the cells were scanned using a CanoScan 8800F (Canon, Tokyo, Japan).

The cell cycle and apoptosis were measured using flow cytometry as previously described (20). For the cell cycle measurements, SW480 and HT-29 cells stably expressing ZNF545 or pcDNA 3.1 were digested and washed twice using precooled phosphate-buffered saline (PBS), then fixed with ice-cold 70% ethanol, stored at 4°C overnight, and then stained with propidium iodide (PI), followed by analysis using CellQuest software (BD Biosciences, San Jose, CA, USA). After the stably transfected cells were collected and washed twice with PBS, they were stained with Annexin V-FITC/PI in the dark, and the cell apoptosis was measured using CellQuest software, followed by data analyses.

Stably ZNF545- or vector-transfected SW480 or HT-29 cells were seeded into 6-well plates, and when the cells reached 95% confluency, a sterile P-20 pipette was used to scratch wounds across each well, followed by removal of cell debris using PBS. The cells were then cultured with medium without FBS to minimize cell proliferation. The images of the wound closure areas of SW480 cells were observed at 0, 48, and 60 h, and the images of the wound closure areas of HT-29 cells were observed at 0, 24, 48, and 84 h using a light microscope (CTR4000; Leica, Germany).

A total of 40 mg of protein lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad). The primary antibodies were against cleaved caspase-3 (12742; Cell Signaling Technology, Danvers, MA, USA), cleaved poly(ADP-ribose) polymerase (PARP) (9541; Cell Signaling Technology), BAX (5023; Cell Signaling Technology), ZNF545 (sc-102235; Santa Cruz Biotechnology, Santa Cruz, CA, USA), PI3K (ab125571; Abcam, Cambridge, UK), phospho-PI3K (Abcam), Akt (4691; Cell Signaling Technology), phospho-Akt (ser473) (4060; Cell Signaling Technology), phospho-GSK3β (ser9) (9323; Cell Signaling Technology), β-catenin (2677; Cell Signaling), active β-catenin (4270; Cell Signaling), c-Myc (1472-1; Epitomic), cyclin D1 (2261; Epitomics), β-actin (LK-ab008-100; Liankebio, China), and Flag (F3165; Sigma-Aldrich).

Six groups of 4-week-old nude mice were used. Empty vector, stably transfected SW480 cells (3×10⁶ cells suspended in 200 µl of PBS) were injected subcutaneously into the left dorsal flank of nude mice, and ZNF545-transfected SW480 cells (3×10⁶ cells suspended in 200 µl of PBS) were injected subcutaneously into the right dorsal flank. After the tumor mass appeared, its length and width were measured every 3 days using a microcaliper. As previously described (23), the tumor volume (mm³) was calculated using the equation: volume = length × width²/2. The mice were euthanized and the tumors were removed when the tumor length reached 1.5 cm. All tumor tissues were weighed before fixation in 4% paraformaldehyde and embedded in paraffin, followed by preparation of the sections for immunohistochemical analyses. All experimental procedures were approved by the Animal Ethics Committee of the Experimental Animal Center of the Chongqing Medical University, Chongqing, China.

The samples of excised tumor tissues from nude mice were fixed in 4% paraformaldehyde before being embedded in paraffin, followed by sectioning into 4-µm slices. After the sections were dewaxed for 2 h in a 60°C incubator, dewaxing and alcohol dehydration were performed in the following order: xylene I, xylene II, xylene III, xylene IV, absolute ethyl alcohol, 95% ethyl alcohol, 80% ethyl alcohol, and 70% ethyl alcohol. Antigen retrieval was performed by boiling the slides in citrate buffer solution for 20 min, followed by cooling at room temperature. The sections were then incubated in 3% hydrogen peroxide for 20 min to prevent endogenous peroxidase activity and washed three times with PBS, then incubated in sheep serum for 20 min. The sections were incubated at 4°C overnight with rabbit anti-PCNA monoclonal antibody. The next day, the sections were warmed to room temperature for 2 h, then washed three times with PBS and incubated with rabbit secondary antibody for 20 min. The slices were washed three times with PBS and incubated with horseradish peroxidase-labeled streptomycin anti-biotin antibody for 20 min. The sections were washed three times, followed by color development with DAB. The sections were washed with water, and the cell nuclei were dyed using hematoxylin, followed by observation using light microscopy.

All statistical analyses were performed using SPSS statistical software for Windows, version 16. Student's t-test was used to compare the expression of ZNF545 in CRC tissues and adjacent normal tissues. The four table Chi-square test or the t-test were used to analyze categorical variables. A value of p<0.05 was considered statistically significant.

The messenger RNA (mRNA) expression of ZNF545 in CRC and its corresponding adjacent tissues was detected by quantitative PCR. The results showed that the expression of ZNF545 was downregulated in tumor tissues compared with the adjacent tissue (p<0.05, Fig. 1A). RT-PCR was then used to examine the expression of ZNF545 in the HT-29, SW480, HCT116, CoCo-2, and LoVo colorectal cell lines. ZNF545 expression was downregulated or silenced in all cell lines compared with the vector (Fig. 2A). In addition, analyses of an online microarray database (www.oncomine.org, Compendia Bioscience, Ann Arbor, MI, USA) showed that ZNF545 was downregulated in CRC tissues but not in normal adult colorectal tissues (p<0.001; Fig. 1B and C). Prognostic analyses from the PrognoScan database (http://www.prognoscan.org/) showed that patients with higher expression of ZNF545 mRNA had significantly increased relapse-free survival compared with those with lower ZNF545 mRNA levels (Fig. 1D). Taken together, these results suggested that ZNF545 was a cancer suppressor gene.

Aberrant promoter methylation is a common mechanism involved in tumor suppressor gene silencing in cancers (21,22). To identify whether the decreased expression of ZNF545 was due to promoter methylation, we determined the methylation status of the ZNF545 promoter in five CRC cell lines using MSP. The results showed that ZNF545 was methylated in all cell lines that were associated with silenced or reduced ZNF545 expression, while normal colorectal tissues expressed significantly higher levels of ZNF545 mRNA (Fig. 2A). We then treated SW480 and HT-29 cells which have low-expression of ZNF545 with Aza, TSA and combination, results showed that its expression was restored together with increasing unmethylated alleles of the ZNF545 promoter (Fig. 2B). The results suggested that promoter methylation is directly responsible for ZNF545 downregulation in colorectal cancer cells.

To investigate whether ZNF545 promoter methylation existed in CRC tissues, MSP was used to examine 32 primary colorectal carcinomas and six normal colorectal tissues. The clinical information of colorectal carcinoma tissues are shown in Table II. We observed that ZNF545 methylation was detected in 28 out of 32 (87.5%) CRC tissues, but in only two out of six (33.33%) normal colorectal tissues (Fig. 2C and D and Table III p=0.0116). Nine pairs of primary CRC tissues and para-carcinoma tissues which were randomly selected in the 32 CRC tissues, were also tested for ZNF545 expression by qPCR. Most tumors have lower ZNF545 mRNA expression compared with their paired adjacent tissues (Fig. 2E), while displaying a higher level of promoter methylation.

To determine whether ZNF545 is a functional tumor suppressor gene in CRC we used the MTS assay and the colony formation assay to measure the growth-inhibiting effects of overexpressed ZNF545 in SW480 and HT-29 cells. The transfection efficiencies of our ZNF545 constructs were confirmed by RT-PCR and western blotting of tumor cell lines and stable cell lines that were selected, respectively (Fig. 3A and B). The MTS cell proliferation assay showed that the cell viability of the ZNF545-overexpressing cells significantly decreased at 48 and 72 h compared with cells transfected with the empty vector (p<0.05; Fig. 3C). The colony formation assay showed that the colony number of cells transfected with ZNF545 was reduced by ~45% when compared with that transfected with the empty vector in SW480 and HT-29 cells (p<0.001; Fig. 3D), indicating that expression of ZNF545 decreased the cloning efficiency. Together, these results showed that ZNF545 inhibited cell proliferation in CRC.

Further studies characterized the effects of ZNF545 on apoptosis and the cell cycle of CRC cells. Fig. 4A shows that the stably-transfected SW480 and HT-29 cells showed a significantly increased number of cells accumulating in the G0/G1 phase when compared with control cells (p<0.01), suggesting that the inhibition of cell proliferation by ZNF545 was likely mediated by a cell cycle arrest at the G0/G1 interface. In addition, flow cytometry showed that the percentage of apoptotic Annexin V-PI-positive cells increased with the overexpression of ZNF545 (Fig. 4B and C).

We also determined the apoptosis of CRC cell lines by western blot analyses of Bax, the active form of caspase-3, and PARP. Fig. 4D shows the increased expression of Bax, cleaved-caspase-3, and cleaved-PARP in both cell lines when compared with cells transfected with the control vector. Together, these results suggested that ZNF545 inhibited cell proliferation by apoptosis and by mediating the cell cycle arrest at the G0/G1 interface.

A wound-healing assay was performed to test the effect of ZNF545 on CRC cell migration. Cells expressing ZNF545 spread along the wound edges significantly more slowly than the control cells (Fig. 5), suggesting that ZNF545 suppressed the migration of SW480 and HT-29 cells.

We also determined if ZNF545 could suppress the growth of CRC cells in vivo. SW480 cells stably transfected with ZNF545 or an empty vector formed tumors in nude mice (Fig. 6A). The subcutaneous tumor growth curve of SW480 cells stably-transfected with ZNF545 or with empty vector showed a significant difference (p<0.05; Fig. 6B), and the tumor volumes from the ZNF545-treated animals were significantly smaller than that of the control group (p<0.05; Fig. 6C). Moreover, the mean weight of the tumors formed by ZNF545-transfected cells was significantly lower than that of tumors formed from the empty vector control cells (p<0.05; Fig. 6D), suggesting that ZNF545 inhibited the growth of CRC cells in vivo. After using PCNA to stain the tissue sections from nude mice, immunohistochemical analyses showed that cell proliferation in the tumors from empty vector-treated cells was greater than that of tumors from the ZNF545-treated animals (Fig. 6E).

Multiple zinc finger family members have been reported to interact the β-catenin activity, and we investigated whether ZNF545 functions as a tumor suppressor through Wnt/β-catenin signaling pathway. The results showed that the expression of active-catenin was downregulated. Moreover, we characterized the effects of ZNF545 on the PI3K/Akt and ERK signaling pathways. The results showed that phospho-AKT and phospho-ERK1/2 were downregulated, indicating that the two pathways also participated in the carcinogenic process. GSK3β is a downstream target gene of the PI3K/AKT pathway and ZNF545 resulted in a profound reduction of GSK3β (Fig. 7A).