Download SNCG shRNA suppressed breast cancer cell xenograft formation

SNCG shRNA suppressed breast cancer cell xenograft formation and
growth in nude mice
SHEN Peihong1, FAN Qingxia 2,*, LI Yanwei3, ZHANG Wei4, HE Xiaokai2, WANG
Zhen4 and ZHANG Yunhan2
1
The First Affiliated Hospital, and Department of Pathology, The Fifth Affiliated
Hospital, Zhengzhou University, Zhengzhou 450000, China
2
Department of Oncology, the First Affiliated Hospital, Zhengzhou University,
Zhengzhou 450051, China
3
Department of Pathology, The Fifth Affiliated Hospital, Zhengzhou University,
Zhengzhou 450052, China
4
Graduate School, Zhengzhou University, Zhengzhou 450001, China
*Corresponding author: Prof. Fan Qingxia, Department of Oncology, The First
Affiliated Hospital, Zhengzhou University Tel.: +86-0371-66902152; Email:
[email protected]
Grant support: A Specified Key Research Grant from Henan Province (No.
92102310224).
Abstract
Background: Overexpression of breast cancer-specific gene 1 (SNCG) is associated
with poor prognosis in advanced breast cancer patients. This study aimed to determine
the effects of SNCG knockdown in breast cancer cells by using small hairpin
(sh)RNA. Methods: Four different SNCG shRNA oligonucleotides were designed
and chemically synthesized to construct mammalian expression vectors. These vectors
were then stably transfected into a breast cancer MCF-7 cell line to knockdown
SNCG expression. After SNCG knockdown was confirmed, the stable cell lines were
inoculated into nude mice. SNCG mRNA and protein expressions were analyzed by
semi-quantitative RT-PCR and immunohistochemistry, respectively in both the stable
cell lines and xenografts. Results: All four SNCG shRNA constructs significantly
reduced SNCG mRNA and protein levels in MCF-7 cells, as compared to the
unrelated sequence control shRNA and the liposome control mice (P < 0.05).
SNCG-knockdown MCF-7 cells formed significantly smaller tumor masses than cells
expressing the unrelated sequence control or the liposome control mice (P < 0.05).
Conclusions: SNCG shRNA effectively suppressed breast cancer cell formation in
vivo and may be a useful clinical strategy to control breast cancer.
Keywords: Breast cancer, SNCG, shRNA, MCF-7 cells, xenograft assay; RT-PCR
INTRODUCTION
Breast cancer is the leading cause of cancer-related death in women between the ages
of 35 and 45 years old and represents the second most common type of cancer and the
fifth most common cause of cancer death in the world. Although recent diagnostic
advances have enabled the identification of breast cancer at very early stages,
remarkably improving treatment outcomes, many women still develop metastatic
disease and ultimately die[1]. Development of breast cancer is thought to involve
multiple genetic alterations, including activation of oncogenes and inactivation of
tumor suppressor genes[2]. The oncogenic protein-encoding breast cancer-specific
gene 1 (SNCG) has been found to be overexpressed in advanced breast cancers.
Subsequent study determined that overexpression of SNCG is an independently
predictive biomarker for breast cancer tumor recurrence and metastasis; thus, SNCG
has been hypothesized as a potential therapeutic target for breast cancer[3].
SNCG is a member of the synuclein family of proteins that are believed to be
involved in pathogenesis of neurodegenerative diseases. Synucleins are small soluble
proteins expressed primarily in neural tissue and certain tumors[3]. The three
synucleins that comprise the entire protein family (-synuclein, -synuclein, and
SNCG, the latter is known as -synuclein) are characterized by a highly conserved
alpha-helical lipid-binding motif with similarity to the class-A2 lipid-binding domains
of the exchangeable apolipoproteins. However, the normal exact cellular function of
-synuclein remains to be defined, although some data have suggested a role in the
regulation of membrane stability and/or turnover[3].
A previous study by cancer researchers revealed that SNCG was completely absent in
normal mammary gland tissue and in the early stages of breast cancer, but was highly
expressed in advanced breast cancer[4]. Another study found that in colorectal cancer
tissues aberrant expression and demethylation of SNCG was correlated with disease
progression and a sensitive predictor of lymph node involvement[5]. Still other studies
have reported that SNCG expression levels are significantly associated with
perineural invasion/distant metastasis and are an effective prognostic factor in
pancreatic cancer[3-6].
Tobacco exposure was able induce altered expression of SNCG in lung cancer cells
through down-regulation of a key methyltransferase gene, DNMT3b. In addition, the
gene that encodes the AP-1 transcription factor has been proposed as a key regulator
SNCG gene expression, since inhibition of AP1 transactivation led to down-regulated
SNCG expression and suppressed the tumor phenotype. Furthermore, -synuclein was
implicated in late stage breast and ovarian cancer metastasis by enhancing cell
motility through activation of the RHO family small-GTPases and ERK1/2[4]. SNCG
was also required for effective ER-alpha signaling in breast cancer[5]. Collectively,
these findings suggest that SNCG could be a potential target for tumor treatment[3-5].
In this study, we investigated whether knockdown of SNCG expression using small
hairpin (sh)RNA could effectively control breast cancer.
MATERIALS AND METHODS
Materials
A human breast cancer MCF-7 cell line was obtained from Nanjing Kenou Biology
Co., Ltd. (Nanjing, China). RPMT-1640 cell growth medium and Hyclone fetal
bovine serum (FBS) were purchased from Baoxin Bio-Technology Co., Ltd.
(Zhengzhou, China). The RNA isolation reagent was from Shanghai GenePharma
Company (Shanghai, China). pGPU6 expression vector carrying SNCG shRNAs,
nonsense shRNA, and vector-control and RNAi-Mate transfection reagents were
custom-made by Shanghai Jima Medicine and Technology Co., Ltd. (Shanghai,
China). Nude mice were purchased from the Institute of Laboratory Animal Slack Co.,
Ltd. (Shanghai, China). The reverse transcription-polymerase chain reaction (RT-PCR)
kit, SNCG primers, and molecular weight markers were purchased from Baosight
Biotechnology Co., Ltd. (Zhengzhou, China). Anti-SNCG antibody was obtained
from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). The SP hypersensitivity
kit for immunohistochemistry was obtained from Beijing Biosynthesis Biotechnology
Co., Ltd. (Beijing, China).
Cell culture
MCF-7 cells were grown in RPMT-1640 containing 10% FBS, penicillin (100 u/ml),
streptomycin (100 u/ml) in a humidified incubator at 37oC with 5% CO2.
Construction of SNCG shRNA and gene transfection
pGPU6 vectors carrying one of four different SNCG shRNA constructs were
custom-designed by using online software available from the Ambion and TaKaRa
SNCG shRNA sequences were:
shRNA2,
shRNA1, 5'-CCAAGGAGAATGTTGTACAGA-3';
5'-CAAGACCAAGGAGAATGTTGT-3';
5'-GCCAAGACCAAGGAGAATGTT-3';
5'-TGGTGAGCAGCGTCAACACTG-3'.
shRNA3,
and
A
nonsense
shRNA4,
sequence
shRNA
(5'-GTTCTCCGAACGTGTCACGT-3') was prepared and inserted into the pGPU6
vector
for
use
as
a
negative
control;
GAPDH
shRNA
(5'-
GTATGACAACAGCCTCAAG-3') was constructed as a positive control. All
plasmids were synthesized and constructed by Shanghai GenePharma Company
(China). The resultant plasmids were amplified and confirmed by sequencing prior to
use. pGPU6 vector containing the different SNCG shRNA constructs, vector-only,
nonsense, and positive control vectors were transfected into MCF-7 cells using a
transfection kit from Shanghai Jima Medicine and Technology Co., Ltd. (China)
according to the manufacturer’s instructions. Forty-eight hours later, the cells were
treated with 800 g/mL of G418. Two weeks later, the G418-resistant clones were
selected and expanded in 400 g/mL G418-containing growth medium. Knockdown
of SNCG expression was confirmed by RT-PCR and immunocytochemistry.
Nude mouse xenograft assay
The animal care and usage protocol was carried out with approval from our
Institution’s Animal Care and Ethics Committee. SNCG shRNA and control
vector-transfected breast cancer MCF-7 cells were grown in monolayer in
RPMT-1640 plus 400 g/mL G418 for three days and then subcutaneously injected
into nu/nu nude mice (8 weeks of age) in the right flank through a 22-gauge needle
with 2  106 tumor cells mixed with 50% Matrigel (BD Biosciences, Bedford, MA,
USA) for a total volume of 200 l per mouse. The animals were then monitored for
tumor formation and growth. At the end of the experiments, the tumor xenografts
were excised for wet-weight determination and analysis.
RT-PCR
SNCG mRNA levels were analyzed by using a semi-quantitative RT-PCR. Briefly,
total cellular RNA was isolated from MCF-7 xenografts using Trizol reagent and then
reversely transcribed into cDNA using a reverse transcription kit. cDNA was then
amplified in a mixture containing 2 l cDNA, 1 l primer each, 2 l dNTP, 0.5 l Taq,
2.5 10 x buffer under the following conditions: initial denaturation at 94oC for 4 min;
35 cycles of 94oC for 30s, 54oC for 45s, 72oC for 1 min; and a final extension at 72oC
for 10 min. The PCR products were separated on a 1.5% agarose gel by
electrophoresis and visualized under a UV light.
Immunohistochemistry
The xenografts taken from the nude mice were embedded in paraffin blocks and
sectioned for immunostaining with an hypersensitivity immunohistochemistry kit.
Briefly, the sections were de-waxed in xylene twice for 10 min each, rehydrated in a
gradient ethanol series (100% down to 50%), and then incubated with 3% H2O2 at
37oC for 10 min and heated at 95oC for 15 min in 0.01M citrate buffer (pH 6.0) for
antigen retrieval. After that, the sections were incubated with normal goat serum at
37oC for 10 min and replaced with the first antibody and incubated overnight at 4oC.
The next day, the sections were washed with PBS three times for 5 min each and then
incubated with a secondary anti-biotin antibody at 37oC for 30 min. Following
washing with PBS, the sections were incubated with horseradish peroxidase-labeled
streptomycin avidin solution at 37oC for 30 min. For color development, the sections
were incubated with 3'-diaminobenzidine/H2O2 solution for up to 10 min. After
washing with tap water, the sections were counterstained with hematoxylin solution
and then subjected to conventional dehydration in ethanol (50% up to 100%) and
“clarification” in xylene and mounting with coverslips.
Control sections were
incubated with normal goat IgG instead of primary antibodies or with the second
antibody only. The stained sections were reviewed and scored using a Nikon
microscope to visualize cytoplamic and nuclear staining of SNCG protein. Five high
power fields were randomly selected and analyzed by using a high-definition[4].
Statistical analysis
All measurements, such as SNCG expression in xenografts, are presented as mean ±
standard deviation (SD) and statistically analyzed by using a paired Student’s t-test
with SPSS software, version 10.0 (SPSS Inc., Chicago, USA). Statistically significant
values were defined as P < 0.05.
RESULTS
Detection of SNCG shRNA transfection efficiency
Transfection of SNCG shRNA vector into MCF-7 cells resulted in about 5-10%
efficiency, as assessed under a fluorescent microscope. We, therefore, made stable
MCF-7 cell sub-lines for each of the four SNCG shRNA constructs, the vector-only,
and the nonsense shRNA vector for subsequent injections into nude mice to generate
xenografts.
SNCG mRNA expression in mouse xenografts
The levels of SNCG mRNA in MCF-7 cells of the SNCG shRNA-transfected MCF-7
cells were assessed by semi-quantitative RT-PCR analysis. Levels of SNCG mRNA
were found to be much lower in SNCG shRNA-transfected MCF-7 cells than in the
liposome or non-sense control groups (P﹤0.05). There was no significant difference
among the levels detected for the four SNCG shRNA-transfected groups (P > 0.05) or
between the non-sense control and the liposome control groups (P > 0.05) (Table 1
and Histogram 1).
Table 1. Levels of SNCG mRNA expression in MCF-7 cells
Transfected construct
SNCG mRNA
concentration
(mean ± SD)
SNCG-siRNA-1
0.624 ± 0.010*
SNCG-siRNA-2
0.626 ± 0.013*
SNCG-siRNA-3
0.634 ± 0.008*
SNCG-siRNA-4
0.631 ± 0.010*
Nonsense shRNA
0.976 ± 0.076#
Liposome control
0.983 ± 0.052#
*P < 0.05 vs. control; #P > 0.05 between the two different controls
SNCG protein expression in MCF-7 cells
SNCG protein expression was assessed in the transfected MCF-7 cells by using
immunocytochemical analysis. Expression of SNCG protein was found to be
significantly decreased in MCF-7 cells transfected with any of the four SNCG shRNA,
as compared to the controls (Fig. 1 and Histogram 2). Quantification of the protein
levels in immunocytochemically stained sections (Table 2) revealed SNCG was
significantly reduced by SNCG shRNA in all four SNCG shRNA-sublines, as
compared to the control groups (P < 0.05).
Table 2. Expression of SNCG protein in MCF-7 cells
Transfected construct
SNCG protein
concentration
(mean ± SD)
SNCG-siRNA-1
4.27 ± 0.12*
SNCG-siRNA-2
4.19 ± 0.22*
SNCG-siRNA-3
4.15 ± 0.14*
SNCG-siRNA-4
4.17 ± 0.13*
Nonsense shRNA
7.92 ± 0.22#
Liposome control
8.02±0.13#
*P < 0.05 vs. control; #P > 0.05 between the two different controls
SNCG shRNA-mediated inhibition of xenograft formation and growth in vivo
The respective MCF-7 sublines were subcutaneously injected into nude mice (n=5 per
group). After 14 days of growth, the tumor masses obtained from the SNCG
shRNA-transfected MCF-7 cell xenografts were remarkably smaller than those from
the control mice (P﹤0.05). There was no difference in tumor mass wet-weights from
any four of the different SNCG shRNAs (P > 0.05) (Table 3).
Table 3. SNCG shRNA inhibition of breast cancer cell xenograft formation and
growth
Xenograft
Group
(×10-3g)a
% of inhibitionb
Liposome control
12.23 ±0. 679
0
Nonsense shRNA
11.60 ± 0.52
0.54 ± 0.236
SNCG -shRNA1
2.01 ±0. 08
8.80 ± 0.32
SNCG -shRNA2
2.14 ±0. 14
8.69 ± 0.24
SNCG -shRNA3
2.03 ±0. 09
8.79 ± 0.27
SNCG -shRNA4
2.10 ± 0.14
8.73 ± 0.19
a
F = 986.134, P < 0.001
b
F = 1750.94, P < 0.001 (% of inhibition); *% of inhibition = [(Liposome control –
shRNA)/ liposome control]
SNCG mRNA expression in mouse xenografts
The levels of SNCG mRNA in mouse xenografts of the SNCG shRNA-transfected
MCF-7 cells were assessed by semi-quantitative RT-PCR analysis. The levels of
SNCG mRNA were much lower in SNCG shRNA-transfected tumor xenografts than
in either the vector or nonsense control tumors (Fig. 2 and Table 4) (P < 0.05). There
was no significant difference among the four SNCG shRNA-transfected groups (P >
0.05) or between the nonsense control and the normal control tumors (P > 0.05).
Table 4. Levels of SNCG mRNA expression in breast cancer cell xenografts
Group
Levels of SNCG mRNA
(mean ± SD)
Liposome control
2.62 ± 0.68
Nonsense shRNA
2.19 ± 0.37
SNCG-shRNA1
1.28 ± 0.36
SNCG-shRNA1
1.28 ± 0.36
SNCG-shRNA3
1.21 ± 0.47
SNCG-shRNA4
0.86 ± 0.17
F = 12.492, P < 0.001, SNCG shRNA vs. the control
SNCG protein expression in mouse xenografts
Immunohistochemical analysis was performed to assess the levels of SNCG protein
expression in the xenografts. The xenografts from all four SNCG shRNA-transfected
MCF-7 sublines presented expression of SNCG protein that was significantly
decreased from the levels detected in the controls (Fig. 3). Furthermore, SNCG
protein levels were significantly reduced by SNCG shRNA in all four of the SNCG
shRNA xenografts, as compared to the control groups (P < 0.05) (Table 5).
Table 5. SNCG protein expression in breast cancer cell mouse xenografts
Gene transfection
SNCG protein levels
(mean ± SD)
Nonsense shRNA
16.56 ± 0.86
Liposome control
17.90 ± 1.38
SNCG-shRNA1
8.06 ± 0.21
SNCG-shRNA2
6.98 ± 0.43
SNCG-shRNA3
6.98 ± 0.72
SNCG-shRNA4
7.70 ± 0.47
F=215.479, P < 0.001,
SNCG shRNA vs. the control
DISCUSSION
In the current study, we have determined the anti-tumor growth effects of SNCG
knockdown using SNCG shRNA in vivo. In particular, we found that SNCG shRNA
significantly reduced expression of SNCG mRNA and protein in mouse xenografts, as
compared to controls. Furthermore, tumor masses were dramatically decreased in
response to knockdown of SNCG expression. The data from the current study
indicates the potential benefit of SNCG shRNA as a clinical strategy to control breast
cancer. Future studies will aim to determine the underlying molecular mechanism of
this phenomenon and evaluate the feasibility of patient safety and effectiveness.
SNCG is a human gene ocalized at 10q23.20-23.3. Its cDNA is approximately 5 kb in
length and comprised of five exons that translate into a protein of 127 amino acids[5,6].
Under physiological conditions, SNCG protein expression is localized to nervous
tissues[7-9]; however, an analysis of disease-specific cDNA library[10] found SNCG to
be highly expressed in the cancerous states of the mammary glands, liver, stomach,
and pancreas, especially at advanced tumor stages. Collectively, the data suggested
that SNCG may play a role in tumor progression and metastasis, and implicated it as a
tumor marker. Since then, many studies have demonstrated that those metastatic
breast and ovarian cancers that overexpress SNCG are particularly insensitive to
certain chemotherapeutic drugs, suggesting that SNCG might be exploited as a
therapeutic target[14-17].
RNA interference is an established and effective means to specifically knockdown
gene expression, and is a promising strategy by which to accomplish gene therapy.
RNAi is a sequence-specific double-stranded (dsRNA) RNA that is capable of
degrading the homologous mRNA[18], resulting in specific gene silencing at the
translational level. RNAi is generally accepted as being more effective than antisense
technology. Therefore, RNAi or shRNA may be novel therapeutic tools for treatment
of many diseases[19-21]. Many previous studies, using shRNA to such common
cancer-associated factors as VEGF, WT1, and KDR, have demonstrated the
usefulness of RNAi; all of which effectively inhibited tumor formation and growth in
nude mouse xenograft models of breast cancer MCF7 cells[22-25]. Likewise, our data
indicates that SNCG shRNA is capable of inhibiting tumor formation and growth in
nude mice.
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