Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 289-296
doi:10.1111/j.1745-7270.2008.00405.x
Zinc finger
protein 278, a potential oncogene in human
colorectal cancer
Xiaoqing Tian, Danfeng Sun,
Yanjie Zhang, Shuliang Zhao, Hua Xiong, and Jingyuan Fang*
Shanghai
Jiaotong University School of Medicine, Renji Hospital, Shanghai Institute of
Digestive Diseases, Shanghai 200001, China
Received: December
13, 2007
Accepted: February
12, 2008
*Corresponding
author: Tel, 86-21-63200874; Fax, 86-21-63266027; E-mail,
This
work was supported by a grant from the National Basic Research Program of China
973 program (No. 2005CB522400)
Zinc finger
protein 278 (ZNF278) is a novel Krueppel Cys2-His2-type zinc finger protein
that is ubiquitously distributed in human tissues. Whether ZNF278 is related to
the development of colorectal cancer is still unclear. The transcriptional
level of ZNF278 was studied in colorectal cancer by real-time polymerase
chain reaction. The results showed that ZNF278 expression was increased in 53%
of colorectal cancer tissues compared to corresponding non-cancerous tissues.
The transcriptional down-regulation of ZNF278 was detected in only three
(6%) human colorectal cancer tissues compared to corresponding non-cancer
tissues. No significant difference was detected in 19 (41%) pairs of samples.
However, we failed to find a significant association between the up-regulation
of ZNF278 transcription and age, sex, the degree of infiltration, or the
tumor size of colorectal cancer. To study the function of ZNF278 in colorectal
carcinogenesis, the colon cancer cell line SW1116 was stably transfected with a
wild-type ZNF278 plasmid to construct an overexpression system, and was transiently
transfected with the small interfering RNA of ZNF278 to construct a ZNF278
knockdown system. Cell proliferation was assessed with
3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide dye and a cell
counter. The results show that ZNF278 promotes cell growth, and its knockdown
suppresses cell proliferation. ZNF278 could be a potential proto-oncogene in
colorectal cancer.
Keywords ZNF278; proto-oncogene; colorectal cancer; cell cycle; cell
proliferation
Colorectal cancer has traditionally been one of the most common
malignancies in Europe and North America, whereas cancers of the upper
gastrointestinal tract and liver have predominated in Asian populations [1].
However, during the past few decades, there has been a remarkable rise in the
incidence of colorectal cancer in Asia [2]. Colorectal carcinomas arise through
aberrant expression of some oncogenes and tumor suppressor genes [3,4].
Identification of novel cancer-related genes will contribute to the understanding
of the mechanism of colorectal carcinogenesis. The zinc finger protein gene family is large, and 1% of all human
genes could belong to this superfamily [5]. Among this family, the
Cys2-His2(C2H2) subtype is the largest subfamily, including approximately 700
proteins [6]. Zinc finger proteins have important physiological functions in
cell proliferation and differentiation [7–10]. Their aberrant
expression is related to various diseases, including cancers [11–15]. Zinc finger protein 278 (ZNF278), also named POZ/BTB and
AT-hook-containing zinc finger protein (PATZ), is a recently identified
transcription factor with seven C2H2-type zinc fingers [16]. ZNF278 belongs to
the Krueppel C2H2-type zinc finger protein family [16]. It is a novel zinc
finger protein that is ubiquitously distributed in human tissues. Although the
physiological role of ZNF278 is not clear, experimental evidence suggests that
it is a potential transcription repressor [16,17]. In small round cell sarcoma,
this gene is fused to Ewing sarcoma (EWS) gene by a small inversion in 22q; the
hybrid is then thought to be translocated t(1;22)(p36.1;q12). The rearrangement
of chromosome 22 involves intron 8 of EWS and exon 1 of this gene, thus
creating a chimeric sequence containing the transactivation domain of EWS fused
to the zinc finger domain of this protein [18]. However, whether ZNF278 is
related to other primary cancers is still unknown. The aim of this study was to
detect the expression level of ZNF278 in human colorectal cancer
samples, and study the basic function of ZNF278 in a human colorectal
cancer cell line.
Materials and Methods
Tissue samples and cell line
Forty-seven colorectal cancer tissues were obtained from patients
undergoing surgery before chemotherapy at Renji Hospital, Shanghai Jiao Tong
University School of Medicine, Shanghai, China, in compliance with our
Institutional Review Board. From each patient, we obtained adjacent tumor-free
parenchyma from a region located 5 cm from the tumor to serve as a paired
control. Immediately after surgical removal, tissue samples were snap-frozen in
liquid nitrogen then maintained at –80 ?C until use. Colon cancer cell line SW1116
cells were maintained in RPMI 1640 medium (Gibco BRL, Gaithersburg, USA)
supplemented with 10% fetal bovine serum (Gibco BRL) under 5% CO2 humidified atmosphere and at 37 ?C as previously described [19].
Real-time RT-PCR for ZNF278 mRNA expression
Total RNA was isolated using TRIzol reagent according to the manufacturer’s
instructions (Invitrogen/Gibco BRL, Carlsbad, USA). RT reactions were carried
out using 5 mg total RNA in a final reaction volume of 20 ml and
Superscript II reverse transcriptase (Invitrogen). Relative quantitation data
were obtained using the comparative Ct method with the ABI PRISM 7700 Sequence
Detection System (software version 1.6; ABI, Foster City, USA) according to the
manufacturer’s protocol. The primers for ZNF278 were: F,
5-GCAGACACAGCACGGAGAT-3; and R, 5-CGCTGAACACCGACTCAAAGT-3. Real-time PCR was
also carried out using the primers for b-actin to normalize each of
the extracts for amplifiable human RNA. The results were expressed as the ratio
of copies of target genes to b-actin. The Ct values were measured, and the average Ct of the triplicate
samples was calculated. Significant alteration in mRNA expression was defined
as a 3-fold difference in the expression level between cancer tissues and
adjacent non-cancerous tissues.
Construction of expression
vectors and stable transfection
To construct the wild-type ZNF278 (GenBank accession No. NM_032050)
expression vector, a PCR-generated full-length ZNF278 cDNA was inserted into
the EcoRI-HindIII sites of the expression vector
pcDNA3.1/Myc-histone A (kindly gifted by Dr. Xiaoqing Chen, Shanghai Jiaotong
University, Shanghai, China). The plasmid, pcDNA3.1-ZNF278, was confirmed by
DNA sequence analysis. Nested PCR was carried out to amplify the full-length
ZNF278 cDNA. The following primers were used: F1, 5-CGGCGCACCTGCGAGACTACAGA-3
and R1, 5-TCCCAGCAGTCCCCAGATGGTTGT-3 for the first PCR; and F2,
5-CCCAAGCTTCCATGGAGCGGGTGAAC-3 and R2, 5-CCGGAATTCTTTCCCTTCAGGCCCCAT-3
for the second PCR. Before transfection, 5?105 SW1116 cells were seeded in 6 cm wells. The
cells were transfected with 1 mg of either pcDNA3.1-ZNF278 or pcDNA3.1 using Effectene
Transfection Reagent (Qiagen, Hilden, Germany), in accordance with the
manufacturer?
instructions. After 24 h, the medium was replaced with a fresh medium. The
cells were further incubated for 24 h, and the medium was replaced with that
containing 300 mg/ml G418 for approximately 30 d, and the medium was replaced every
day. Overexpression of ZNF278 was confirmed by real-time RT-PCR and Western
blot analysis using anti-c-Myc tag sequence antibody (Sigma, St. Louis, USA).
RNA interference and transient
transfections
ZNF278 small interfering RNA (siRNA) (sense,
5-GCGCCGAUAUAAUGCUCUUTT-3 and antisense, 5-AAGAGCAUUAUAUCGGCGCGG-3) and
negative control siRNA (sense, 5-UUCUCCGAACGUGUCACGUTT-3 and antisense, 5-ACGUGACACGUUCGGAGAATT-3)
were designed and synthesized (Shanghai GenePharma, Shanghai, China).
Mock-transfected or pcDNA3.1-ZNF278-transfected SW1116 cells were transfected
using 80 nM of each siRNA duplex. The siRNA was complexed with the transfection
reagent in a serum- and antibiotic-free medium for 8 h. After applying the
transfection reagents, the cellular medium was replaced with the
serum-containing maintenance medium, and the cells were incubated for 48 h.
Selective silencing of ZNF278 was confirmed by real-time RT-PCR and Western
blot analysis using anti-c-Myc tag sequence antibody (Sigma).
Cell viability assay
Cell viability assay
Cell growth was assessed using
3-(4,5-dimethylthiazol-2-l)-2,5-diphenyltetrazolium bromide (Sigma) with an
absorption maxima at 570 nm, according to the manufacturer’s instructions.
Briefly, 5?103 cells stably transfected with pcDNA3.1-ZNF278 or pcDNA3.1 were
seeded per well in a 96-well flat-bottom plate. The cells were allowed to grow
for 48 h, and 20 ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (5
mg/ml in phosphate-buffered saline) was then added to each well. After 3 h
incubation at 37 ?C, the cells were lysed by the addition of 0.1 M HCl in
isopropanol alcohol; this produced a color, the absorbance of which was monitored
at 570 nm. In addition, 5?103 SW1116 cells were transfected in complete medium containing 80 nM
of siRNA-ZNF278 or siRNA-negative control for 48 h, and cell viability was then
assayed.
Cell proliferation studies
Cells in the log growth phase (7?104) stably transfected with
either pcDNA3.1-ZNF278 or pcDNA3.1 plasmid were seeded in a 24-well flat-bottom
plate for the assessment of in vitro cell growth. These cells were
trypsinized and counted on a Casy Counter (Schaerfe System, Reutlingen,
Germany) for 6 d. The SW1116 cells (5?104) were transfected with 80 nM siRNA-ZNF278 or siRNA-negative
control, and the cell number was counted at 48 h after transfection.
Flow cytometry for the detection of cell cycle progression
Cell cycle analysis was carried out by flow cytometry. Approximately
1?106 cells were removed and
washed twice with phosphate-buffered saline and fixed in ice-cold ethanol for 1
h. The samples were then concentrated by removal of ethanol and exposure to 1%
(V/V) Triton X-100 (Sigma) and 0.01% RNase (Sigma) for 10 min at
37 ?C. Cellular DNA was stained in the dark with 0.05% propidium iodide for 20
min at 4 ?C. Cell cycle distributions were determined using a flow cytometer
(FACSCalibur; Becton Dickinson, San Jose, USA). The data obtained from 10,000
cells were analyzed using the MultiCycle software package (Phoenix Flow
Systems, San Diego, USA).
Statistical analysis
Data are representative of at least three independent experiments
carried out in triplicate, and are presented as the mean±SD. Comparisons
between groups were made using Student’s t-test. The data for tissue
sample groups were compared using the Sign test. Relationships were analyzed by
Fisher’s exact test using SAS 6.12 software (SAS Institute, Cary, USA). A value
of P<0.05 was taken to indicate a significant difference between the mean values.
Results
ZNF278 expression is
up-regulated in human colorectal cancer tissue
Real-time quantitative PCR was carried out to evaluate the amounts
of ZNF278 mRNA in colorectal cancer samples (n=47) and the
corresponding non-cancerous samples (n=47). ZNF278 transcription was
found to be significantly up-regulated in 25 (53%) human colorectal cancer
tissues compared to the corresponding non-cancer tissues (c2=15.75, P<0.05) (Fig. 1). The transcriptional
down-regulation of ZNF278 was detected in three (6%) human colorectal
cancer tissues compared to the corresponding non-cancerous tissues (Fig. 1).
No significant difference was detected in 19 (41%) colorectal cancer tissues
and the corresponding non-cancerous tissues (Fig. 1). There were no
significant association in the up-regulation of ZNF278 expression and age, sex,
the degree of infiltration, or tumor size of colorectal cancer (data not
shown).
ZNF278 functions as a
potential proto-oncogene in colorectal cancer
To assess the function of ZNF278 in colorectal cancer, we cloned
human ZNF278 cDNA in the expression vector pcDNA3.1/Myc-histone A. The siRNA
was transiently transfected in order to knock down the ZNF278 expression.
Overexpression of ZNF278 in the stable selected transfectants and knockdown of
ZNF278 by siRNA transfection were confirmed by real-time RT-PCR and Western
blot analysis (Fig. 2). We first examined the proliferative effects of
ZNF278 on the colorectal cancer cells.
3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assays showed that
the absorbance of the pcDNA3.1-ZNF278-transfected cells was 0.84±0.06, and that
of the pcDNA3.1-transfected cells was 0.58±0.03. The absorbance of the siRNA-ZNF278-transfected
cells was 0.67±0.03, whereas that of siRNA-negative control-transfected cells
was 0.88±0.02. Student’s t-test indicated a significant difference
between the groups (P<0.01). In addition, the cell growth curve showed that the in vitro tumor cell growth was significantly promoted in
the cells transfected with the ZNF278 plasmid as compared to control cells [Fig.
3(A), P<0.01]. Cell counting assay showed that the numbers of the cells transfected with siRNA-ZNF278 and siRNA-negative control were (4.66±0.35)?105 and (5.50±0.53)?105, respectively. Knockdown of
ZNF278 significantly inhibited cell growth [Fig. 3(B), P<0.01]. Our results indicated that transfection of the SW1116 cells with pcDNA3.1-ZNF278 promoted cell growth, and siRNA transfection resulted in a significant inhibition of cell growth (P<0.01).To further study the function of ZNF278 on the cell cycle, we
evaluated the cell cycle of the cells stably transfected with pcDNA3.1-ZNF278
or pcDNA3.1 and of the cells transiently transfected with siRNA-ZNF278 or
siRNA-negative control. As shown in Table 1, the overexpression of
ZNF278 in the cells transfected with the pcDNA3.1-ZNF278 plasmid significantly
increased the percentage of the S-phase cells and decreased the percentage of
the G0/G1-phase cells (P<0.05) [Fig. 4(A,B)]. The knockdown of ZNF278 expression significantly blocked
the cell cycle at the G0/G1 phase (P<0.05) [Fig. 4(C,D)].
Discussion
Colorectal cancer is a common malignant tumor worldwide, with the
incidence increasing in Asian countries [2]. Aberrant gene expression is
involved in colorectal carcinogenesis [3,4]. The zinc finger domain is a typical feature of zinc finger proteins.
It consists of several cysteine and histidine residues, and the fold is created
by the binding of specific amino acids in the protein to a zinc atom [20]. Many
zinc finger proteins belong to the C2H2-type zinc finger protein family
[21,22]. Zinc finger protein possibly targets the gene promoter region and
regulates gene expression [23,24]. Zinc finger proteins have important
physiological functions in human development and differentiation. For example,
Egr-1 controls cell proliferation and apoptosis [25]. Aberrant expression of
zinc finger proteins is related to various diseases, including cancers [11–15]. For
example, aberrant expression of KLF6 and ST18 is associated with
hepatocellular carcinoma and breast cancer, respectively [12,14]. ZNF278 is a
novel zinc finger protein and might function as a transcription repressor
[16,17]. Therefore, whether it is involved in carcinogenesis is an interesting
topic for study.The ZNF278 protein contains an AT-hook DNA-binding motif that
usually binds to other DNA-binding structures to play an important role in
chromatin modeling and transcription regulation [16,17]. Its Poz domain is
thought to function as a site for protein-protein interaction and is required
for transcriptional repression [16,17]. ZNF278 belongs to the C2H2-type zinc
finger protein family. Some studies have supported that C2H2-type zinc finger
proteins regulate cell proliferation, growth, differentiation, and
carcinogenesis [9,10,26,27]. The ZNF278 protein has typical features of a
transcription factor. It was suggested to be a transcription repressor [16,17].
Based on this research, ZNF278 might be considered to be an important factor in
the physiological state. Aberrant expression of ZNF278 could lead to disease.
In one published report, the rearrangement of the ZNF278 gene was
detected in small round cell sarcoma [18]. However, it is still unknown whether
ZNF278 is related to other primary cancers.In the present study, we examined the ZNF278 expression level in
colorectal cancer tissues and corresponding non-cancerous tissues, and found
that the ZNF278 expression was significantly higher in cancer tissues than in
the non-cancerous tissues. This suggested that the up-regulation of ZNF278
expression might contribute to colorectal tumor carcinogenesis. In particular,
ZNF278 is a type of zinc finger protein and contains domains involved in
DNA-binding and protein-protein interactions. It is possible that ZNF278 is
involved in some important signaling pathways or regulates the transcription of
other important genes. However, we failed to find an association between the
ZNF278 expression level and age, sex, the degree of infiltration, or tumor size
of colorectal cancer. Possibly, ZNF278 influences the initiation but not the
progression of colorectal cancer. This inference has to be verified in the
future. In addition, because of absence of specific antibodies against ZNF278,
protein expression levels under biological state could not be analyzed by
Western blot or immunohistochemical methods. It will be useful to study the
expression of ZNF278 protein in tumor tissues in the future. In order to identify the function of ZNF278, we constructed a
wild-type ZNF278 expression vector and transfected the SW1116 cells. In
addition, we transiently transfected the SW1116 cells with ZNF278 siRNA. We
studied the effect of ZNF278 on the biological function of the cells with
regard to the overexpression and knockdown of ZNF278. The results of our study
revealed that ZNF278 promoted colorectal cancer cell growth, and that the knockdown
of ZNF278 suppressed cell growth and arrested the cell cycle. According to the
abovementioned results, the function of ZNF278 is similar to that of other
proto-oncogenes such as c-myc [28]. ZNF278 could be a potential
proto-oncogene in colorectal carcinoma. Unfortunately, the knockdown of ZNF278
did not induce apoptosis (data not shown). It is likely that ZNF278 does not
influence the signal pathway of apoptosis.In summary, the up-regulation of ZNF278 expression was
observed in human colorectal cancer tissues. ZNF278 might be a potential
oncogene in colorectal cancer.
Acknowledgements
We thank Dr. Xiaoqing Chen (Shanghai
Jiaotong University, Shanghai, China) for providing the plasmid
pcDNA3.1/Myc-histone A, and Mrs. Hongyin Zhu, Mrs. Weiqi Gu, and Mr. Enling Li
in our laboratory for their technical support.
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