Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 519-525
doi:10.1111/j.1745-7270.2008.00424.x
Knockdown of STAT3 by shRNA inhibits the growth of CAOV3 ovarian
cancer cell line in vitro and in vivo
Feng Huang1#*, Xiaoyun Tong2#, Liangqing Fu3, and Ronghua Zhang1
1 Department of Pharmacology, College of
Pharmacy, Jinan University, Guangzhou 510632, China
2 Department of Cardiology, The First
Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine,
Guangzhou 510405, China
3 Department of Clinical Pharmacology, 307
Hospital, Beijing 100850, China
Received: February
21, 2008
Accepted: April 14,
2008
This work was
supported by a grant from the Natural Science Foundation of Jinan University
(JN200301)
#
These
authors contributed equally to this work
*Corresponding
author: Tel, 86-20-33091767; Fax, 86-20-85220850; E-mail, [email protected]
Constitutively activated signal transducer
and activator of transcription 3 (STAT3) plays an important role in the formation
of many tumors including ovarian cancer. In this study, RNA interference
specific to STAT3 was employed to study its effects on the inhibition of STAT3
signaling and on the growth of ovarian cancer CAOV3 cells. Plasmid vectors
pGenesil-1-GFP-U6 expressing specific small hairpin RNA (shRNA) against STAT3
and the scrambled shRNA control were constructed. After transfection into CAOV3
cells, the STAT3 shRNA specifically suppressed STAT3 expression at both mRNA
and protein levels. At the same time, expressions of Bcl-xL, cyclin D1, and c-myc
were down-regulated, whereas the cleaved caspase 3 was up-regulated. In
addition, STAT3 knockdown inhibited anchorage-independent growth and induced
apoptosis in CAOV3 cells, and decreased tumor growth in nude mice implanted
with ovarian cancer cells.
Keywords CAOV3; STAT3; siRNA; cell
proliferation; apoptosis
Ovarian cancer is one of the leading causes of death in women with
gynecological neoplasms [1]. To develop new strategies for treatment, it is pivotal
for us to gain a better understanding of the molecular mechanisms of the proliferation
and differentiation of ovarian cancer cells. Signal transducers and activators of transcription (STATs)
constitute a family of transcription factors involved in cellular responses to
cytokines and growth factors. Previous studies revealed that the STAT pathway
plays important roles in several malignant human cancers including ovarian
cancer [2,3], and activated STAT signaling is involved in cell growth and differentiation.
Accordingly, the disregulation in the activation of STATs is associated with
malignant transformation [4]. Constitutive activation of STAT3 has been
reported in a variety of cancer cells such as breast carcinoma, lymphoma,
prostate cancer, and ovarian cancer cells. As STAT3 activation promotes
tumorigenesis through its effects on cell proliferation, differentiation, and
anti-apoptosis [2,3,5], the STAT3 signaling pathway is a potential target for
tumor therapy. RNA interference (RNAi) is a relatively new technology that has been
used for sequence-specific gene silencing. It is mediated by short interfering
RNA (siRNA) molecules produced from long double-stranded RNA (dsRNA) by the
enzymatic activity of Dicer in cells. RNA interference can be experimentally
achieved by delivery of a synthetic dsRNA or a plasmid DNA vector containing
sequence coding for a small hairpin RNA (shRNA) [6].In the present study, we showed that vector expressing
STAT3-specific shRNA knocked down the expression of STAT3 in transfected CAOV3
ovarian cancer cells, and inhibited cell growth in vitro and in vivo,
which might be correlated with the down-regulation of Bcl-xL, cyclin D1, and c-myc,
and the up-regulation of the cleaved caspase 3 in these cells.
Materials and Methods
shRNA preparation and plasmid constructionThe pGenesil-1-GFP-U6 plasmid from Genesil Biotechnology (Wuhan,
China) was used for DNA vector-based shRNA construction. Based on the cDNA
sequence of STAT3 (GenBank accession No. NM_003150) and the shRNA designing
tool provided freely by Ambion at its website (http://www.ambion.com/techlib/misc/siRNA_finder.html),
we synthesized DNA templates encoding one STAT3-specific shRNA and one
scrambled missense control (Sangon Biological Engineering Technology and
Services, Shanghai, China). The sequences of oligonucleotides encoding STAT3
shRNA were: 5?–gatccCATCTGCCTAGATCGGCTAttcaagacgTAGCCGATCTAGGCAGATGttttttGTCGACA-3? (upper strand)
and 5?–agcttgtcgacaaaaaaCATCTGCCTAGATCGGCTAcgtcttgaaTAGCCGATCTAGGCAGATGG-3? (lower strand).
The sequences encoding the shRNA control were scrambled from the above STAT3
DNA: 5?–gatccCTGCATAGACCTGCTATCGttcaagacgCGATAGCAGGTCTATGCAGttttttGTCGACA-3? (upper strand),
and 5?–agcttgtcgacaaaaaaCTGCATAGACCTGCTATCGcgtcttgaaCGATAGCAGGTCTATGCAGG-3? (lower strand).
These sequences are short hairpin DNAs containing a sense strand of 19
nucleotides followed by a short spacer (TTCAAGACG) and six Ts that act as the
transcriptional stop signal. The oligonucleotide pairs were designed to form
artificial enzymatic overhangs for BamHI, SalI, or HindIII
at their terminals, and thus can be directly ligated into pGenesil-1-GFP-U6
after annealing. After ligation and transformation into bacteria, the positive colonies
were selected by antibiotic resistance. The correct sequences of the final DNA
preparations were confirmed by sequencing (Sangon).
Cell culture and transfection The human ovarian carcinoma cell line CAOV3 was purchased from the China
Center for Type Culture Collection (Wuhan, China) and maintained in Dulbeccos
modified Eagles medium (DMEM) (Gibco Life Technologies, Paisley, UK)
supplemented with 10% fetal bovine serum (Gibco), 100 U/ml penicillin, and 100 mg/ml
streptomycin at 37 ?C in a 5% CO2 incubator. Cells were detached
with trypsin-EDTA solution. After washing, they were resuspended in fresh
medium and used for subsequent experiments. For transfection, the CAOV3 cells
were transfected with the plasmids with Lipofectamine reagent (Invitrogen,
Carlsbad, USA) according to the manufacturers instructions (in our system, the
percentage of cells transfected with siRNA was approximately 75%). In all of
the following experiments, whenever the shRNA plasmid transfected cells were used,
mock transfection without any DNA and negative control transfection with shRNA
missense control plasmid were always included as experimental controls.
Reverse transcription-polymerase chain reaction (RT-PCR)Total RNA was extracted from cells with Trizol reagent (Invitrogen)
72 h after transfection. Two micrograms of total RNA was subjected to reverse
transcription with an RT-PCR kit (Promega, Madison, USA). The primers (Sangon)
used for subsequent PCR amplifications were: STAT3, 5?-TTGCCAGTTGTGGTGATC-3? (forward) and 5?-CAGACCCAGAAGGAGAAGC-3? (reverse) for
amplification of a 318 bp product; c-myc, 5?-GGGCTTCTCAGAGGCTTGGC-3? (forward) and 5?-CGTCCTTGCTCGGGTGTTGTA-3? (reverse) for
amplification of a 341 bp product; and b-actin, 5?-ATGTTTGAGACCTTCAACAC-3? (forward) and 5?-CACGTCACACTTCATGATGG-3? (reverse) for
the amplification of a 491 bp product. Amplification conditions were 95 ?C for
5 min, then 30 cycles of 94 ?C for 30 s, 55 ?C for 30 s, and 72 ?C for 40 s,
followed by the 72 ?C extension for 3 min.
Cell proliferation assay The viability of CAOV3 cells was determined with a
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
inner salt (MTS) assay kit (Promega). Cells were plated in a 96-well cell
culture plate at the density of 10,000 cells/well, five wells/group, then
transfected as described above. Seventy-two hours after transfection, the novel
tetrazolium compound MTS was added to each well with culture medium and
incubated for 2 h. The absorbance of each well was measured at 490 nm with a
96-well plate reader (680 microplate reader; BioRad, Hercules, USA).
Anchorage-independent growth assayAnchorage-independent growth of tumor cells was assayed by their
ability to form colonies in agar plates. Forty-eight hours after transfection,
CAOV3 cells were trypsinized and suspended in DMEM with 0.3% agar at a density
of 600 cells/ml, and seeded into a 6-well plate at 1 ml/well, five wells/group.
After incubation in regular culture medium at 37 ?C with 5% CO2 for 2 weeks, the cells were stained with 0.3% crystal violet and
colonies larger than 100 mm in diameter were counted.
Determination of DNA fragmentation Apoptosis was assayed with a cell death detection enzyme-linked
immunosorbent assay kit (Roche Diagnostics, Lewes, UK) designed to detect
apoptosis-induced DNA fragmentation. The transfected cells were cultured for 3
d and the fragmented DNAs from these cells were measured according to the
manufacturers instructions.
Flow cytometry analysis Distribution of cells at different stages of the cell cycle was
analyzed by flow cytometry. Approximately 1?105 cells were fixed by PBS with 75% ethanol at 4
?C for 40 min, and incubated with 40 ng/ml propidium iodide (Sigma, St. Louis,
USA), 0.1 mg/ml RNase, and 0.1% Triton X-100, at room temperature for 40 min,
then analyzed by flow cytometry (Becton Dickinson Immunocytometry Systems, San
Jose, USA) for DNA contents.
Western blot analysisCells (5?105) were washed and lysed in 100 ml of lysis buffer [20 mM
HEPES (pH 7.3), 1% Triton X-100, 10% glycerol, 50 mM NaF, 0.1 mM EDTA, 10 mg/ml
phenylmethylsulphonyl fluoride, 1 mM Na3VO4, and 5 mg/ml leupeptin] on ice for 30 min. Lysates were centrifuged at
10,000 g at 4 ?C for 10 min. Approximately 15 ml or 40 mg of total
protein in the supernatant was subjected to SDS-PAGE and reacted with
anti-STAT3, anti-phosphorylated-STAT3 (p-STAT3; Ser727), anti-Bcl-xL,
anti-cleaved caspase 3 (Asp175), and anti-cyclin D1 antibodies (Cell Signaling
Technology, Beverly, USA). Anti-b-actin antibody from Santa Cruz Biotechnology
(Santa Cruz, USA) was used as the Western blot loading control.
Tumor growth in vivo The male nude mice (5–6-week-old) were maintained in a
temperature-controlled environment with free access to standard rodent chow and
water. Experiments were carried out in accordance with the ethical guidelines
of Guangzhou University of Traditional Chinese Medicine (Guangzhou, China).
Equal numbers of CAOV3 cells transfected with STAT3 or scrambled STAT3 were
collected by trypsinization 3 d after transfection. The cells were washed three
times with 1?PBS and resuspended in
0.1 ml saline solution. Each mouse was injected with 5?106 cells to form a tumor
xenograft and eight mice were used in each transfected group. Then STAT3 siRNA
or scrambled STAT3 vector diluted in saline were directly injected into the
tumors at a dose of 20 mg every 7 d. The tumor volumes were assessed once every week for a
total of 6 weeks by measuring the two perpendicular dimensions with a caliper
according to the formula: volume=(a?b2)/2,
where a is the larger and b is the smaller dimension of the
tumor.
Statistical analysis The data obtained were statistically evaluated using one-way ANOVA
followed by Bonferronis test. Differences in values with P<0.05 were
considered statistically significant.
Results
CAOV3 cells were transfected with DMEM
alone (mock), or the plasmid expressing STAT3 scrambled shRNA (scrambled
vector) or the plasmid expressing STAT3-targeted shRNA (STAT3 vector). At 72 h after
transfection, they were then harvested for the extraction of total RNA or
proteins, or subjected for further analysis on their growth capacities and
their apoptosis, as described in detail below.
Efficient knockdown of STAT3 by STAT3-specific shRNA The STAT3 mRNA levels in transfected cells were measured by
semi-quantitative RT-PCR, and the protein levels for STAT3 and p-STAT3 were
determined by Western blot analysis. As shown in Fig. 1(A), STAT3
mRNA expression was significantly suppressed in CAOV3 cells transfected with
the STAT3 shRNA expressing vector. Accordingly, the protein levels for both
STAT3 and its activated form, phosphorylated-STAT3 [Fig. 1(B)] were also
markedly reduced in the same cells. In mock transfection or the scrambled shRNA
control, cells showed no significant changes in the levels of STAT3 mRNA or
protein. In addition, as shown in Fig. 1(C), treatment of CAOV3 cells
with STAT3 shRNA caused a time-dependent inhibitory effect on STAT3 expression
in CAOV3 cells.
Inhibition of cancer cell proliferation by STAT3 knockdown To examine the effect of STAT3 knockdown on proliferation of CAOV3
cells, the MTS kit was used to measure proliferation status of the cells. Fig.
2 indicates that treatment of CAOV3 cells with STAT3-targeted shRNA
resulted in approximately 50% growth inhibition of CAOV3 cells, whereas no
significant inhibition was observed in the cells transfected with the STAT3
scrambled vector compared with the mock transfected cells.
Inhibition of anchorage-independent growth by STAT3 knockdown Anchorage-independent growth in 3-D agar gel is a measure of
cellular transformation, one of the main characteristics of cancer cells. When
transfected cells were plated into 3% agar plates and grown for 2 weeks, we
observed a significant decrease of approximately 62% in their ability to form
colonies for STAT3 knockdown CAOV3 cells compared with both controls,
indicating a reduced transforming ability (Fig. 3).
Induction of apoptosis by STAT3 knockdown Apoptosis induced by STAT3 shRNA in CAOV3 was examined by following
the kinetics of DNA fragmentation with a nucleosomal fragment detection
enzyme-linked immunosorbent assay kit. STAT3 knockdown significantly increased
DNA fragmentation compared with the controls [Fig. 4(A)]. DNA content
analysis by flow cytometry showed that STAT3 shRNA obviously increased the
fraction of cells at the sub-G1 phase of the cell cycle [Fig.
4 (B)]. In addition, cleaved caspase 3 was detected only in the cells
treated with STAT3 shRNA by Western blot analysis [Fig. 4(C)], but not
in any of the control cells.
Down-regulation of Bcl-xL, cyclin D1, and c-myc by STAT3
knockdown Previous studies have shown that constitutive activation of STAT3
induces the expression of Bcl-xL, cyclin D1, and c-myc, which might
contribute to tumorigenesis. We investigated the potential effects of STAT3
knockdown on these STAT3 downstream molecules. Western blot analysis detected
the specific bands for Bcl-xL and cyclin D1 individually in transfected CAOV3
cells. As shown in Fig. 5(A), the protein levels of Bcl-xL and cyclin D1
were significantly reduced in STAT3 knockdown CAOV3 cells. RT-PCR analysis
revealed that c-myc mRNA was also decreased [Fig. 5(B)] in the
same cells.
Inhibition of in vivo tumor growth by STAT3 knockdown To investigate the potential effect of STAT3 knockdown on the growth
of ovarian cancer cells in vivo, ovarian tumor xenografts were produced
in nude mice with STAT3 knockdown CAOV3 cells or the controls. Growth curves of
these xenografts over 6 weeks showed that the STAT3 shRNA group displayed a
significantly lower growth rate, only approximately 50% of that obtained from
the two control groups (Fig. 6).
Discussion
STAT3 exists in a latent form in the cytoplasm and can be activated
by a range of signaling pathways starting from the activation of many cell
surface receptors by tyrosine phosphorylation. This mechanism of STAT3
activation has been shown to be involved in the elevated levels of cell
proliferation, anti-apoptotic effects, and cell cycle progression in many
cancer cells [7]. STAT3 protein contains a tetramerization and a leucine zipper
domain at its N-terminus, a DNA-binding domain in the middle, and an SH2
transactivation domain at the C-terminus [8]. Upstream regulators of STATs,
such as Janus family tyrosine kinases (JAKs), steroid receptor co-activator
(Src) and epidermal growth factor (EGF), regulate STAT3 by phosphorylation at
Tyr705, which in turn results in the activation of a cascade of STAT3-mediated
signaling events [9]. Under physiological conditions, STAT3 activation is
transient and lasts from several minutes to several hours, so aberrantly
sustained activation of STAT3 could lead to uncontrolled growth and prolonged
survival often seen in cancer cells [10].
RNAi technology is widely used in gene therapy and as a research
tool for gene modulations because it allows the selective, transient knockdown
of targeted protein expression. In mammalian cells, RNAi can be triggered by
siRNAs that cause strong yet transient inhibition of the expression of specific
genes. This could be achieved by delivering a synthetic double-stranded siRNA
or a plasmid vector containing DNA sequence coding for an shRNA. Chemically
synthesized siRNAs can be delivered to the cytosol and directly combined with
RNAi silencing complex, resulting in effective suppression of gene expression.
However, such suppression is transient, as the oligos are easily degraded by
the host cell. The expression of shRNA from plasmid has some advantages as
shRNA plasmids can repeatedly transcribe similar dsRNA products and suppress
gene expression over a longer period of time. This plasmid vector-based
approach is also less expensive compared with the direct delivery of the
synthesized siRNA [11,12]. shRNA vectors have been shown to efficiently block
expression of specific proteins both in vitro and in vivo, and
these artificial shRNAs are transcribed from the U6 or H1 promoters in the
targeting vectors [13]. The effect of STAT3 knockdown in ovarian cancer has not been well
studied. Here, we showed that transfection into CAOV3 cells with STAT3 shRNA
vector dramatically inhibited the expression of STAT3 mRNA as well as protein,
and also decreased cell proliferation and anchorage-independent colony
formation. Combined analyses of DNA fragmentation, cell cycle progression by
flow cytometry, and cleaved caspase 3 expression by Western blot analysis
indicated that STAT3 knockdown induced apoptosis of CAOV3 cells. Importantly,
STAT3 shRNA also significantly suppressed tumor cell growth in vivo. Previous studies showed that constitutively activated STAT3 led to
up-regulation of its downstream molecules such as anti-apoptotic protein Bcl-2
and cell cycle regulators cyclin D1 and c-myc, all playing important
roles in oncogenesis [14,15]. Consistent with these reports, our study showed
that CAOV3 cells with STAT3 knockdown decreased their expression of Bcl-xL
protein, as well as the expression of cyclin D1 and c-myc [Fig. 5(A)].
This might imply that STAT3 plays a role in the regulation of these molecules
in ovarian cancer cells, and its knockdown results in growth inhibition and
induction of apoptosis. In summary, our study showed that knocking down STAT3 with shRNA
produced from pGenesil-1-GFP-U6 led to growth inhibition and apoptosis of CAOV3
cells in vitro and inhibition of tumor growth in vivo. Therefore,
it is tempting to speculate that STAT3 signaling might play a considerable role
in ovarian cancer, in addition to its known roles in many other cancers.
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