High expression of Zinc-finger protein X-linked promotes tumor growth and predicts a poor outcome for stage II/III colorectal cancer patients

Zinc-finger protein X-linked (ZFX) was recently identified as a novel oncoprotein in several human malignancies. In this study, we examined the correlation between ZFX expression and the clinical characteristics of stage II/III CRC patients, as well as the molecular mechanism by which ZFX apparently contributes to CRC tumor progression. Using immunohistochemistry, we detected expression of ZFX in CRC tissues collected from stage II/III patients and determined that its expression correlated with tumor differentiation and stage. Survival analysis indicated that patients with high ZFX expression had poorer overall and disease-free survival. ZFX knockdown in SW620 and SW480 CRC cells significantly inhibited cell proliferation and colony formation, enhanced apoptosis and induced cell cycle arrest. It also enhanced the sensitivity of CRC cells to 5-Fu. In a xenograft model, ZFX knockdown suppressed in vivo CRC tumor growth. Microarray analysis revealed the primary target of ZFX to be DUSP5. Whereas ZFX knockdown increased DUSP5 expression, DUSP5 knockdown rescued ZFX-mediated cell proliferation in ZFX knockdown cells. These findings demonstrate that ZFX promotes CRC progression by suppressing DUSP5 expression and suggest that ZFX is a novel prognostic biomarker and potentially useful therapeutic target in stage II/III CRC patients.


Cell culture and RNA interference
Each human CRC cell line (HCT116, SW480, Caco-2, LoVo, HT-29 and SW620) that was used as part of this study was purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The HCT116 and HT-29 cells were maintained in McCoy's 5A culture medium (Sigma-Aldrich, USA), while the SW480 and SW620 cells were maintained in L-15 culture medium (Gibco, USA). The LoVo and Caco-2 cells were maintained in F12K (Sigma-Aldrich, USA) and MEM (Gibco, USA) culture medium, respectively. All of the cell lines were incubated in a 5% CO2 atmosphere at 37°C.
In order to facilitate stable knock down of endogenous ZFX in CRC cells, three Short hairpin RNAs (shRNA) were designed using the following sequences: 5′-GCCTGAGAATGATCATGGA-3′ (KD1), 5′-CGGAAATTGATCCTTGTAA-3′ (KD2) and 5′-AAT CAGTCTCATTCACATA-3′ (KD3). The negative control shRNA sequence was: 5′-TTCTCCGAACGTGTCAC GT-3′. The synthesized DNA oligonucleotides containing these sequences were then annealed and inserted into the GV118 vector with GFP (Genechem, China). Verification of the inserted sequences was performed using DNA sequencing. To facilitate lentivirus manipulations, the GV118 vector, together with pHelper 1.0 and pHelper 2.0 plasmids (Genechem, China), were transfected into 293 T cells. Forty-eight hours after transfection initiation, the cultured supernatant containing the lentiviral particles was collected and purified. For lentivirus infection, CRC cells that were experiencing logarithmic growth were infected with lentivirus according to the multiplicity of infection (MOI). The lentivirus carried a copy of the Green Fluorescent Protein (GFP) and infection efficiency was assessed based on the numbers of GFP-expressing cells using florescence microscopy. Moreover, commercially generated small interference RNA (siRNA, GenePharma, China) was used to reduce DUSP5 expression in cells and its sequence was designed as follows: 5′-AAGCCAUGACCACAUGGACGA-3′. The sequence of designed negative control was as follows: 5′-UUCUUCGAACGUG UCACGUTT-3′. All the transfection processes were performed using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions.

Quantitative real-time reverse transcription PCR (QRT-PCR)
Total RNA was isolated from cultured cells using Trizol reagent (Invitrogen Life Technologies) according to the manufacturer's instructions. The isolated RNA was www.impactjournals.com/oncotarget/

MTT assay
Both CRC cell groups were seeded into 96-well plates (2500/well for SW620 and 2000/well for SW480) in 200 µl of culture medium. After overnight incubation, 20 μl of MTT reagent (5 mg/L, Beijing Dingguo Changsheng Biotechnology, China) was added into each well. Following incubation at 37°C for 4 hrs, the culture medium was removed and 150 μl of DMSO reagent (Sinopharm Chemical Reagent, China) was added to allow complete dissolution of purple precipitates. After the plates were gently shaken for 10 min, the optical density (OD) of each well was determined using a microplate reader (Biotek, USA) at a wavelength of 490 nm. The absorbance values were used to determine the growth rates of cells, which was calculated as follows: (OD of the sample on the test day)/(OD of the sample at Day 1).

Apoptosis
The apoptosis of CRC cells was measured using the AnnexinV-APC single staining method. Briefly, the cells were washed with D-Hanks solution and were trypsinized. Thereafter, they were collected and centrifuged at 1500 rpm for 5 min and then washed with PBS. Next, after washing with AnnexinV 10 × binding buffer, the cells were incubated with 10 μl AnnexinV-APC reagent (eBioscience, USA) in the dark for 15 min at room temperature. Finally, the cell suspension was transferred to the 96-well plate (200 μl/well) and analyzed by flow cytometry (Millipore, USA).

Cell cycle assay
Each group of cells were washed with D-Hanks solution, digested with trypsin and collected by centrifugation at 1500 rpm for 5 min. After two washes with ice-cold PBS and fixation in 70% ethanol for 1 hr, cells were re-suspended in 1ml staining buffer containing PI (2 mg/ml, Sigma-Aldrich) and RNase A (10 mg/ml, Fermentas, Canada). Finally the cells were subjected to FACSCalibur flow cytometer (BD Biosciences) for cell cycle analysis.

Colony formation assay
Cells in the logarithmic phase were digested, re-suspended and seeded onto the 6-well plates (800/well). After incubation in a 5% CO2 atmosphere at 37°C for two weeks, the cells were washed with PBS and fixed with paraformaldehyde (1 ml/well) for 60 min. Following two washes with PBS, the cells were stained with GIEMSA reagent (500ml/well, Chemicon, Japan) for 20 min. Finally, the colonies were photographed and counted under a fluorescence microscope (Olympus, Japan).

Cytotoxicity assay
SW620 and SW480 cells were seeded onto the 96-well plate (2×104/well) and treated with gradient of 5-FU drug (0.5μg/ml to 1000μg/ml) for 48 hrs. Then cell viability was assessed using the CCK-8 method (10μl/ well, Dojindo, Japan). After incubation for 2 hrs, cells were subjected to a microplate reader for recording an absorbance at a wavelength of 450 nm. The 5-FU sensitivity was evaluated based on IC50, which was defined as the concentration inducing 50% inhibition of cell viability.

Xenograft models
A total of 16 athymic nude male BALB/c mice (4-5 weeks old, 15-18g) were purchased from Shanghai Labarotary, Animal Research Center. The experimental protocol was approved by the Animal Care and Use Committee and the Ethics Committee of the Sixth People's Hospital Affiliated to Shanghai Jiao Tong University. Both type of CRC cells (5.0 × 106 each) in the logarithmic phase were suspended in 200 μL of serum-free medium and then injected subcutaneously into the left hind flank of each mouse. Tumor volumes were monitored every week using a caliper and calculated according to the following formula: volume = ab2/2, where "a" and "b" was defined as length and width of the tumor respectively. Four weeks later, mice were sacrificed, tumors were harvested and tumor weights were measured before sending for immunohistochemical analysis.