Download Knockdown of Rab25 expression by RNAi inhibits growth of human

Pathology (December 2006) 38(6), pp. 561–567
EXPERIMENTAL PATHOLOGY
Knockdown of Rab25 expression by RNAi inhibits growth of human
epithelial ovarian cancer cells in vitro and in vivo
FAN YANG, XIN XIAO-YAN, CHEN BI-LIANG
AND
MA XIANGDONG
Department of Obstetrics and Gynecology, Xijing Hospital Fourth Military Medical University, Xi’an, China
Summary
Aims: Ovarian cancer is the leading cause of cancer death
among gynaecological malignancies. Elevated expression of
Rab25 has been seen in this malignancy. To better understand its role in maintaining the malignant phenotype, we
used RNA interference (RNAi) directed against Rab25 in our
study. RNAi provides a new, reliable method to investigate
gene function and has the potential for gene therapy. The
aim of the study was to examine the anti-tumour effects
elicited by a decrease in the level of Rab25 by RNAi and its
possible mechanism of action.
Methods: According to the Rab25 mRNA sequence in
Genbank, a pair of 64 nt oligonucleotides were designed and
synthesised, each containing the sites of restriction endonuclease at both ends. Oligonucleotides were annealed and
ligated with linearised pSUPER by ligase. The recombinants
(named pSUPER/Rab25 siRNA) were finally sequenced and
identified by enzyme cutting and sequencing. The human
ovarian cell line A2780 was grown without transfection, transfection with empty vector and with pSUPER/Rab25 siRNA with
electroporation. The inhibitory effect was examined by RTPCR, MTT, FCM and tumour growth of athymic nude mice.
Results: Rab25 siRNA expression vector was successfully
constructed and identified by double endonuclease digestion.
Sequence analysis of inserted fragment revealed the same
sequence as synthesised siRNA oligonucleotides. Cells transfected with Rab25 siRNA can specifically knock down the
transcription of Rab25, exhibiting cells with slower proliferation, increased apoptosis, and decreased tumour growth.
Conclusions: Rab25 siRNA expression vector has been
successfully constructed, and it could inhibit the tumour
growth both in vitro and in vivo. Our data suggest that the
Rab25 signalling pathway plays a role in the regulation of cell
proliferation and apoptosis in ovarian cancer cells, which
indicates that the Rab25 gene plays a definite role in the
development and aggressiveness of human ovarian cancer
and should be further elucidated as a possible therapeutic
target of ovarian cancer.
Key words: RNA interference, siRNA, Rab25, ovarian cancer, epithelial
cell.
Received 7 June, revised 30 July, accepted 1 August 2006
INTRODUCTION
Ovarian cancer is a leading cause of death among
gynaecological malignancies. In spite of advances in
surgery and chemotherapy, its mortality rate has remained
relatively unchanged during the past several decades.1
Therefore, an understanding of the molecular mechanisms
involved in ovarian cancer formation and progression
should be helpful in developing more effective treatments
for ovarian cancer.
Emerging evidence implicates alterations in the Rab
small GTPases and their associated regulatory proteins
and effectors in multiple human diseases including cancer.2
Rab proteins are Ras-like small GTPases, which are
involved in regulating various aspects of the membrane
trafficking process.3 More than 60 Rabs are present in
distinct membrane compartments in mammalian cells.4
Studies have recently shown that Rab25, located at
chromosome 1q22, is amplified at the DNA level and
over-expressed at the RNA level in ovarian and breast
cancer. These changes correlated with a worse outcome in
both diseases. In addition, enforced expression of Rab25 in
both breast and ovarian cancer cells decreased apoptosis
and increased proliferation and aggressiveness in vivo,
potentially explaining the worse prognosis.5 A better understanding of genetic alterations as well as the physiological
and pathophysiological roles of Rab GTPases may open
new opportunities for therapeutic intervention and better
outcomes.
Technologies for gene knockout, antisense oligonucleotides and ribozyme have been frequently used to explore
new functions of genes, but their low frequencies have
limited their applications.6 Fortunately, the emergence of
gene ablation technologies based upon the RNA interference (RNAi) phenomenon has provided new opportunities for experimental biology.7,8 Particularly, a generation
of vectors directing the synthesis of short hairpin RNAs
(shRNAs) that are processed to form small interfering
RNAs (siRNAs), enables persistent suppression of endogenous gene expression.9,10 RNA interference (RNAi) can
result in sequence-specific gene silencing. This phenomenon
is now being exploited as a powerful tool for reverse
genetics, and shows great promise for therapeutic applications.11
In the present study, we used RNAi technology to knock
down the expression of the Rab25 gene in human ovarian epithelial cancer cells and to examine their effect in
human ovarian cancer cells in vitro and on tumour growth
in vivo, which may establish candidate status of Rab25mediated signalling for studies in targeted ovarian cancer
therapy.
ISSN 0031-3025 printed/ISSN 1465-3931 # 2006 Royal College of Pathologists of Australasia
DOI: 10.1080/00313020601024037
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YANG et al.
Pathology (2006), 38(6), December
MATERIALS AND METHODS
siRNA design
Three pairs of siRNA were designed according to the human Rab25
sequence in Genbank (BC 009831; Qiagen, Germany) and selected optimal
sites of interfering. As shown in Table 1, each pair contained a unique 19-nt
double-stranded human Rab25 sequence that was presented as an inverted
complementary repeat and separated by a loop of 9 nt spacer. For example:
S1 : 50 GAT CCCCðs -N19ÞTTCAAGA GA(as-N19)TTTTTGGAAA 30 ;
S2 : 30 GGG ðas-N19Þ A AGTTCTCTðs-N19ÞAAAAACCTTTTCGA 50
human Rab25, sense 5’-GGGGAATGGAACTGAGGAAGA-3’ and
antisense 5’-ATTGGTCCGGATGCTGTTCT-3’; for glyceraldehydes-3phosphate dehydrogenase (GAPDH), sense 5’-CAGGGCTGCTTTTAACTCTGG-3’; anti-sense 5’-GTGGACTCCACGACGTACTCA-3’. The
newly synthesised cDNA was amplied by PCR. The reaction mixture
contained 2 mL cDNA template, 1.5 mM MgCl2, 2.5 U Tag polymerase,
and 0.5 mM GAPDH was used as an internal control. Amplification cycles
were: 94uC for 3 min, then 33 cycles at 94uC for 1 min, 58uC for 1 min, 72uC
for 1.5 min, followed by 72uC for 15 min. Aliquots of PCR product were
electrophoresed on 1.5% agarose gels and PCR fragments were visualised
by ethidium bromide (EB) staining. All experiments were repeated on at
least three occasions.
Cell proliferation, MTT assay and flow cytometric analysis
Plasmid construction, transformation and extraction
Oligonucleotides sequence of Rab25 siRNA was chemically synthesised by
TakaRa (Japan). Oligonucleotides were annealed in a buffer containing
10 mM Tris (pH 8.0), 10 mM NaCl and 1 mM EDTA at 95uC for 5 min
followed by 70uC for 10 min. Then ligated into pSUPER vector (Ambion,
USA) that had been cut with Bgl II and Hind III (TakaRa). The plasmids
were then transformed into DH5a competent cells according to the
protocol provided by the manufacturer (TakaRa). After selective culture,
recombined plasmid was extracted from DH5a. The recombined plasmid
was identified via restriction enzyme EcoRI and Hind III (TakaRa) using
guidelines provided by the manufacturer. The positive clones were
confirmed by sequencing (TakaRa). Reactions were carried out in 20 mL
volume containing 2 mL of DNA. Following this, amplification was
performed. Medipreps DNA purification system (Promega, USA) was
used to extract plasmid DNA according to the protocol.
Cell culture and transient transfection
We selected epithelial ovarian cancer cells A2780.5 The ovarian cancer cell
line A2780 was obtained from the laboratory of the Department of
Obstetrics and Gynecology, Xijing Hospital Fourth Military Medical
University, Xi’an, China. This was cultured in RPMI 1640 (Invitrogen,
USA) supplemented with 10% heat inactivated fetal bovine serum, 100 U/
mL penicillin, and 100 mg/mL streptomycin at 37uC in a humidified
atmosphere of 5% CO2 and 95% air.
For transfections, cells were seeded in a 6-well plate at a concentration
of 16106 cells/well and allowed overnight growth to reach 80–90%
confluency.The stable transfections of pSUPER/Rab25 siRNA were
performed in A2780 cells using the Lipofectamine 2000 (Gibco, USA)
following the guidelines provided by the manufacturer. A pure population
of transfected cells can be obtained using a low concentration of G418
(400 mg/mL). Reverse transcriptase polymerase chain reaction (RT-PCR)
analysis or other experiments were performed.
We plated cells at a density of 16105 cells/35-mm dish. Cells were then
cultured in the presence of 10% FBS for 8 days. We harvested and counted
total cells.
Cells were grown in a 96-well plate with a concentration of 26105 and
transfected with plasmids (pSUPER/Rab25 siRNA) using Lipofectamine
2000 (a total of 50 mL). The tetrazolium salt MTT solution (5 mg/mL;
Sigma, USA) was added into each well and incubated at 37uC for 4 h. The
media were carefully aspirated. The blue formazan product converted from
MTT was dissolved by the addition of 150 mL/well dimethyl sulfoxide
(DMSO; Sigma) and optical density (OD) readings were obtained using an
autoreader (Tecan, Austria) at 490 nm. Each experiment was repeated three
times.
Flow cytometry (Beckman-Coulter, USA) analysis was used to
determine apoptosis and cell cycle of the cells. In brief, A2780 cells were
transfected with Rab25 siRNA or without Rab25 siRNA; cells
(56105,16106) were harvested, washed twice with PBS, and resuspended
in 100 mL PBS and stained with propidium iodine (PI; Sigma) for apoptosis
and cell cycle analysis.
Tumour growth
Equal numbers (106,26106) of A2780 cells, without transfection,
transfection empty vector, and transfection with pSUPER/Rab25 siRNA
cells were harvested by trypsinisation, washed twice with 16PBS, and
resuspended in 0.1 mL saline. The suspensions were then injected
subcutaneously into 4- to 6-week-old athymic nude mice. A total of 15
mice was used for each cell line infected with the above three cells. The mice
were kept in pathogen-free environments and checked every 2 days for 1.5
months. The date at which a grossly visible tumour first appeared and the
size of the tumour were recorded. The mice were killed when tumours
reached 2.5 cm in diameter.
Statistical analysis
RNA extraction and RT-PCR analysis
Total RNA was isolated using TRIzol reagent (Invitrogen) according to the
manufacturer’s protocol. The reverse transcription reaction was performed
using the Superscript First-Strand Synthesis System (Invitrogen Life
Technologies, USA) according to the protocol. After incubation at 42uC
for 80 min, the reverse transcription reaction was terminated by heating at
70uC for 15 min. DNA primer sequences were designed as follows: for
TABLE 1 Oligonucleotides sequence of Rab25 specific siRNA
Name
pSUPER/
Rab25siRNA 1
pSUPER/
Rab25siRNA 2
pSUPER/
Rab25siRNA 3
Sequence of siRNA
Target
nucleotides sites
TCCCTCTGGCTGCAGAAGT
75–93 bp
GGTGGTGCTGATCGGCGAA
265–283 bp
TGCCATCACTCTGG GCAGT
787–805 bp
Data are expressed as mean¡SEM. The significance of differences was
determined by ANOVA and post-hoc multiple comparison test using SPSS
11.5 software (SPSS, USA). p,0.05 was considered to be statistically
significant.
RESULTS
Plasmid identification
We designed and synthesised three Rab25 siRNA
sequences, and ligated the sequences into pSUPER vector.
The recombined plasmid (named pSUPER/Rab25 siRNA)
was finally sequenced and identified by enzyme cutting and
sequencing. The result of sequencing confirmed that siRNA
had been ligated to the vector. The transfected vector
extracted was digested with EcoRI and Hind III, then
checked with 1.5 % agarose gel electrophoresis. The length
of the fragments of 291 kb (recombined plasmid) and
227 kb (empty plasmid) showed the success of construction
as expected (Fig. 1).
RNA INTERFERENCE INHIBITS EXPRESSION OF RAB25
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TABLE 2 Decrease in cell proliferation by Rab25 siRNA
A2780
103.5¡7.5
Empty vector
Rab25 siRNA
106.9¡7.4
38.5¡3.5*
*p,0.01.
Cell counts were performed on day 8 in stable cells lines. Numbers
represent cell number 610 000 cells/mL. Each sample is presented¡SEM
of three replicates from one of three representative experiments. Results
were confirmed with multiple subclones of each cell line. Clearly, cells
transfected with Rab25 siRNA showed significantly decreased cell
proliferation and cell counts were significantly less than in the controls.
Fig. 1 Identification of interfering vector by enzyme cutting. 1, DNA
marker; 2, pSUPER/Rab25 siRNA3; 3, empty vector.
The knock-down efficiencies of pSUPER/Rab25 siRNA
in A2780 cells were first evaluated using RT-PCR. After
transfection, relative Rab25 levels were normalised against
mRNA levels of an internal control gene, GAPDH,
performed in the same run. As shown in Fig. 2, cells
transfected with pSUPER/Rab25 siRNA showed a significantly reduced transcription of Rab25 mRNA when
compared with empty vector and cells without transfectants, respectively. In 1–4 months, the suppression of
pSUPER/Rab25 siRNA 3 in the cultured transfection cells
was more powerful than the others.
Further examination was carried out with MTT and flow
cytometry to observe the effect of decreasing Rab25 levels.
Slower proliferation and increased apoptosis in Rab25
silenced cells
Under 10% fetal bovine serum (FBS) conditions, cells with
transfected pSUPER/Rab25 siRNA 3 decreased cell numbers stably (Table 2).
We examined the viability of Rab25 specific siRNA
transfectants. The results showed that RNAi directed
against Rab25 significantly decreased the growth rate of
A2780 cells. As shown in Fig. 3, pSUPER/Rab25 siRNA
transfected A2780 cells began to show a decreased viability
at 48 hours after transfection and the decline slowed down
and became mild at 72 hours. The two control cell lines
grew rigorously with the cell number increased at 48 hours
and the increase proceeding until 72 hours (Fig. 3). The
knock-down of Rab25 in A2780 cells could significantly
inhibit the growth of tumour cells in vitro.
Apoptosis analysis indicated that the number of apoptotic cells increased in A2780 cells transfected with pSUPER/
Rab25 siRNA compared with the controls (Fig. 4).
pSUPER/Rab25 siRNA transfected A2780 cells were most
prone to apoptosis with an apoptotic rate of 12.3%, higher
than the rate of 0.9% in the A2780 control cells. Thus,
treatment with siRNA against Rab25 could activate
apoptotic pathways.
Different cell cycle phases (G1, S or G2/M phase) are
characterised by different DNA contents. Table 3 shows
differences in cell cycle phases in A2780 cells transfected
with or without Rab25 siRNA. It was clear that pSUPER/
Rab25 siRNA transfection induced an increase in the
number of cells in G1 phase and a decrease in the number
of cells in S phase compared with the control (Table 3;
Fig 5).
Decreases in transformation efficiency in vitro and tumour
growth in vivo
Injections of A2780 cells in nude mice demonstrated
relevant tumour biology. In this study, tumour growth
was inhibited by treatment with Rab25 siRNA. Nude mice
were randomly selected for treatment with Rab25 siRNA
which would affect tumour growth in vivo. A2780 cells
transfected with or without the empty vector produced
Fig. 2 RT-PCR analysis. (A) Rab25 and (B) GAPDH. RT-PCR analysis showed a lower mRNA level in A2780 cells transfected with Rab25 siRNA. M,
marker; 1, A2780; 2, empty vector; 3, Rab25 siRNA3.
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YANG et al.
Fig. 3 Growth curve of A2780 cells after Rab25 siRNA transfection. It
can be seen that, when compared with both the empty vector and A2780,
cells transfected with Rab25 siRNA show a remarkable reduction of cell
proliferation, in keeping with the observations of decreased expression of
Rab25 in these transfectants.
tumours 25 mm in diameter within 4 weeks of their
subcutaneous injection into nude mice, while A2780 cells
transfected with Rab25 siRNA showed only minimal
tumour growth at 4 weeks (Fig. 6). Furthermore, Rab25
siRNA signicantly inhibited production of A2780 in the
tumours. Therefore, transfected Rab25 siRNA is a signicantly novel way to inhibit tumour growth in vivo.
DISCUSSION
As far as we know, cancer is caused by abnormalities
in DNA sequence, copy number, rearrangements, or
Pathology (2006), 38(6), December
expression. The accumulation of multiple changes in
critical genes within a single cell is required to escape from
normal controls on cell growth and proliferation, allowing
development into a clinically evident tumour.11 Cancer cells
often show abnormal signal transduction pathways,12
leading to proliferation in response to external signals.13
Oncogene over-expression14 is a common phenomenon in
the development and progression of many human cancers.
Therefore, oncogenes provide a potential target15 for
cancer gene therapy.
We know that Ras proteins are oncogenes and Rab
proteins are Ras-like small GTPases, which have crucial
roles in vesicle trafficking, signal transduction and receptor
recycling, which in turn regulate normal cellular activity.3
Approximately 60 human Rab genes are encoded by the
human genome. However, the complexity of the Rab
protein family might be even greater as there is evidence
that alternative splicing of Rab mRNAs results in the
production of functionally distinct isoforms.16 Recent
studies have shown multiple links between Rab GTPase
dysfunction and associated regulatory proteins in human
diseases, including cancer.17–21
Most Rab proteins are constitutively expressed in all
mammalian cells, although several Rab proteins have been
shown to be differentially expressed in epithelia and
neurons, perhaps fulfilling specialised transport functions
of these polarised cells. Therefore, some Rab protein
functions may be ubiquitous. Rab5 has been localised to
the early endosomal fraction in a number of cell systems.22
Other Rab proteins may have different functions in
Fig. 4 Rab25 gene silencing increases early-stage apoptosis in A2780 cells. A2780 cells were transfected with RabR25 siRNA or empty vector. Cells were
then collected by trypsinisation.The apoptotic cells were determined by flow cytometry. (A) A2780; (B) empty vector; (C) Rab25 siRNA; (D) diagram
summarising the differences among the above groups. A2780 cells transfected with Rab25 siRNA show much greater rates of early apoptosis compared with
the control (p,0.01).
RNA INTERFERENCE INHIBITS EXPRESSION OF RAB25
TABLE 3 Cell cycle phase distribution of A2780 cells after transfection of
Rab25 siRNA
Group
A2780
Empty vector
Rab25 siRNA
G1 %
S%
G2/M %
50.2
48.4
88.6
36.3
37.7
8.1
13.5
14.0
3.3
The cells with Rab25 gene silencing revealed an increase in the percentage
of G1 phase and a decrease in S phase.
different cellular systems. Thus, while Rab2 is associated
with the intermediate endoplasmic reticular-Golgi compartment in Madin–Darby canine kidney (MDCK) cells,23 it is
associated with the H,K-ATPase-containing tubulovesicles
in gastric parietal cells.24 In addition to the ubiquitous
small GTP-binding proteins, some demonstrate tissuespecific distribution, including Rab3A,25,26 Rab3D,27 and
Rabl5.28 Rabl7 is expressed only in the kidney, intestines,
and liver,29 and its distribution suggests a possible role in
transcytosis.
Fig. 5
565
Rab25, which shares 68% sequence identity with
Rab11a, is expressed exclusively in epithelial cells, suggesting it regulates aspects of vesicular transport that are
unique to epithelia.30,31 The sequence of the GTP-binding
site of Rab25 is unique among Rab proteins, in that the P-3
domain consensus sequence WDTAGQE contains a leucine
residue in place of the conserved glutamine. An equivalent
substitution in Ras creates a dominant-active GTPase.32
Casanova et al. have found that Rab25 over-expression
alters transcytosis and apical recycling,33 and Cheng et al.5
have reported that Rab25 controls tumour progression,
aggressiveness, and potentially chemosensitivity, and is
amplified at the DNA level and over-expressed at the RNA
level in ovarian cancer. Rab25 over-expression markedly
increases cancer cell proliferation, preventing apoptosis.
Schaner et al.34 reported similar results and also found that
ovarian cancer patients with elevated Rab25 either failed to
enter a disease-free state following surgery and chemotherapy, or exhibited short durations of disease-free survival.
Therefore, specific down-regulation of Rab25 might be a
potential therapeutic strategy against human ovarian
cancer.
Cell cycle of A2780 cells analysed by flow cytometry. (A) A2780; (B) empty vector; (C) Rab25 siRNA.
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YANG et al.
Pathology (2006), 38(6), December
Fig. 6 Tumour growth in A2780 cells transfected with Rab25 siRNA. (A) Rab25 siRNA significantly inhibits A2780 cell growth in nude mice (p,0.05).
(B) Rab25 siRNA significantly inhibits A2780 cell proliferation in nude mice. We investigated the effects of Rab25 by in vivo subcutaneous injection
into nude mice. Three weeks after transfection there was a tiny tumour in the mouse on the right which received transfected Rab25 siRNA.
Techniques based upon RNAi principle35 have become
powerful and indispensable tools in the molecular toolkit,
being honoured as ‘the breakthrough of 2002’.35,36
By means of the RNAi method, cellular growth assays in
vitro were used to determine the functional consequences of
RNAi-mediated decreases of Rab25 in established ovarian
carcinoma cells. Our results demonstrate that RNAi can
effectively down-regulate Rab25 over-expression with great
specificity and that the plasmids endogenously expressing
siRNA can successfully deplete Rab25 expression in A2780
cells after transfection. Furthermore, the tumour inhibition
effects persist after transfection as shown by experiments in
vitro and in vivo. Therefore, it is not surprising that we have
shown that reduction of the Rab25 level by Rab25 siRNA
can significantly inhibit the growth rate of cancer cells.
Many anticancer agents induce apoptosis. Our data also
suggest that knock-down of Rab25 by Rab25 siRNA in
A2780 cells can increase the sensitivity of these cells to
apoptosis. This was probably one of the reasons for the
anti-tumour effect. Moreover, in cell cycle, increases in G1
and decreases in S further confirm a role for Rab25 in
regulating cellular apoptosis. A recent study has provided
direct evidence that multiple signalling molecules including
AKT, extracellular signal-regulated kinase 1/2, and p38
mitogen-activated protein kinase, associate on endocytic
vesicles and mediate their function by facilitating translocation of the vesicles to the nucleus, implicating Rab
GTPases in conducting cell survival signals.37 However, the
pathways that Rab25 controls and/or that are involved in
the observed apoptosis need further study.
All of the nude mice injected with Rab25 transfected cells
developed subcutaneous tumours. However, mice injected
with Rab25 transfected A2780 cells developed obviously
smaller tumours than the controls, and the controls
developed larger tumours in a shorter time. This further
demonstrates that Rab25 plays a master role in tumour
progression and aggressiveness. These studies have marked
a new target in the genetic manipulation of ovarian cancer
development by allowing Rab25 to be down-regulated by
RNAi.
In conclusion, Rab25 siRNA substantially reduces the
expression of Rab25 mRNA, slows cell proliferation,
increases apoptosis, suppresses transformation, and inhibits tumour growth, demonstrating that Rab25 siRNA
silencing is a powerful approach for dissecting oncogenic
signals in human ovarian cancer. Our data implicate Rab25
in the aggressiveness of ovarian carcinomas suggesting that
Rab25 can be used to predict patient outcome and provide
a novel therapeutic target. These studies also implicate
Rab25 and, by extension, the Rab GTPase family in
tumour aggressiveness. This adds the genes encoding the
Rab family to other members of the Ras oncogene
superfamily as tumorigenic mediators.
Address for correspondence: Dr Y. Fan, Department of Obstetrics and
Gynecology, The Fourth Military Medical University, 17, The West Road,
Xi’an, Xi’an Shaanxi Province, 710032, China.
E-mail: [email protected]
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