Research Paper |
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Acta Biochim Biophys |
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doi:10.1111/j.1745-7270.2005.00077.x |
Silencing of Bcl-XL
Expression in Human MGC-803 Gastric Cancer Cells by siRNA
Xiao-Yong LEI#*, Miao ZHONG#, Lan-Fang FENG, Chun-Yan YAN,
Bing-Yang ZHU, Sheng-Song TANG, and Duan-Fang LIAO
Institute
of Pharmacy and Pharmacology,
Nanhua University, Hengyang 421001, China
Received: January
11, 2005
Accepted: May 12,
2005
This work was supported
by the grants from the National Natural Science Foundation of China (No.
30300426) and the Youth Foundation of Hunan province education department (No.
03B034)
# These authors
contributed equally to this work
*Corresponding
author: Tel, 86-734-8281408; E-mail, [email protected]
Abstract To investigate the inhibitory
effect of the Bcl-XL small interfering RNA (siRNA) on Bcl-XL gene expression
in the human gastric cancer cell line MGC-803, green fluorescent protein (GFP)
siRNA was constructed and transfected into MGC-803 cells, together with GFP
expression vector pTrace SV40. GFP expression levels were observed
using fluorescence microscopy. Bcl-XL siRNA and negative siRNA were then constructed
and stably transfected into MGC-803 cells. RT-PCR and immunofluorescence were
used to detect the expression of Bcl-XL. Spontaneous apoptosis was detected
by acridine orange (AO) and flow cytometry. Results were as follows: (1) 48
h after GFP expression vector and GFP siRNA co-transfection, the expression
level of GFP in the GFP siRNA group was much lower than the negative siRNA
group, according to fluorescence microscopy results. The mRNA and protein
levels of Bcl-XL in Bcl-XL siRNA stable transfectants were reduced
to almost background level compared with negative siRNA transfectants or untreated
cells. (2) Changes in nucleus morphology was observed by AO staining nucleic
and flow cytometry analysis, which showed that stable Bcl-XL siRNA transfectants
have an increased spontaneous apoptosis (21.17%±1.26% vs. 1.19%±0.18%
and 1.56%±0.15% respectively, P<0.05 vs. negative
siRNA or untreated control). siRNA targeting GFP or Bcl-XL genes
can specifically suppress GFP or Bcl-XL expression in MGC-803
cells, and Bcl-XL siRNA can increase spontaneous apoptosis. Bcl-XL siRNA may
be a beneficial agent against human gastric adenocarcinoma.
Key words Bcl-XL; siRNA; MGC-803 cells
Gastric adenocarcinoma is the second leading cause of cancer
mortality in the world and the leading cause of cancer mortality in China.
There is still no effective treatment for patients with advanced gastric
adenocarcinoma [1]. Chemotherapy has generally shown some clinical effect, but
resistance to chemotherapeutical drugs is a big problem. Most of these drugs
act primarily by inducing apoptosis. The development of resistance of cancer
cells to cytotoxic drugs may be a result of resistance to apoptosis. Apoptosis
is regulated in part by the Bcl-2 family including pro-apoptotic Bax and Bak
and anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1. The relative ratio of these
proteins determines the sensitivity or resistance of cells to various
apoptotic stimuli [2,3]. It was reported that most cancers show over-expression
of anti-apoptotic proteins. It may be a good therapeutic method to
down-regulate over-expressed anti-apoptotic genes, such as Bcl-2 and Bcl-XL,
or IAP and Mcl-1 genes. In fact, down-regulation of Bcl-2
expression by antisense oligonucleotides is currently at the final stage of
clinical trial. But antisense oligonucleotide technology also faces many
problems, including low absorption rates, non-specific inhibition effects,
large effective dosage and toxicity [4,5]. Recently, the successful use of
small interfering RNAs (siRNAs) showed a promising therapeutic method. RNA
interference (RNAi) is a cellular pathway of homologous gene silencing in a
sequence-specific manner at the mRNA level. The basic mechanism of RNAi is that
a double-stranded RNA (dsRNA) is broken into short pieces called short interfering
RNA (siRNA), which trigger the activation of an RNA-cutting enzyme
(ribonuclease) directed specifically to degrade just the messenger RNA
related to the trigger by an identical sequence, whereas other genes remain
unaffected [6–8]. RNAi may provide a new
therapeutic technique for tumors such as leukemia, melanoma, breast, colon and
cervical cancer [9–12]. Several studies have
documented that successful down-regulation of BCR-ABL, bcl-2, c-raf
and xIAP expression in human myeloid leukemia cells results in inducing
apoptosis [13–15], and down-regulation of
MDR-1 results in up-regulating chemosensitization of human pancreatic
and gastric cell lines [16].
In this work, we observed the inhibitory effect of siRNA targeting Bcl-XL
on the human gastric cancer cell line MGC-803 and the increased spontaneous
apoptosis in cells transfected with Bcl-XL siRNA.
Materials and Methods
siRNA vector construction
pSilencer 3.1-H1 vector was purchased from Ambion (Austin, USA). The
Bcl-XL siRNA inserting sequence had sense and antisense sequences as follows: Bcl-XL
sense sequence 5‘-CAGGGACAGCATATCAGAG-3‘, antisense sequence 5‘-CTCTGATATGCTGTCCCTG-3‘.
The Ambion web-based target sequence converter was used to convert siRNA target
sites into double-stranded DNA fragments with BamHI and HindIII
sticky ends. The fragments were synthesized by Shanghai Sangon (Shanghai,
China), annealed, and ligated into the linearized pSilencer vector. Negative
control vector that expresses a hairpin siRNA with limited homology to any
known sequences in the human genome and green fluorescent protein gene (GFP)
control insert template were provided with the vector kit.
Cell lines and transfection
Human gastric adenocarcinoma cell line MGC-803, obtained from Sun
Yat-Sen University (Guangzhou, China), was routinely maintained in phenol
red-free Dulbecco’s modified Eagle medium (DMEM; Gibco BRL, Grand Island, USA)
containing 100 ml/ml fetal bovine serum (Hyclone, Logan, USA), 37 ºC in a
humidified atmosphere with 5% CO2 in air. Cells grown in 6-well plates
were transfected with Lipofectamine 2000 and harvested 2 d after the
transfection, then cells were split at a ratio of 1:12 in 24 wells. After 24 h,
geneticin (G418; Amresco, Solon, USA) at the final concentration 400 mg/ml was added
to select transfected Bcl-XL siRNA and negative siRNA cells. The cultures were
refreshed using G418-containing medium every 4 d. After 8 d, colonies were
observed and picked to expand the culture. After 14 d, cells were harvested and
examined. Plasmids encoding GFP and siRNAs were generally used at a
ratio of 1:1. GFP siRNA and negative siRNA cells were directly detected under
fluorescence microscope 48 h after GFP expression vector and GFPsiRNA or
negative siRNA co-transfection.
RT-PCR
Bcl-XL siRNA or negative siRNA stably transfected cells and
untreated control cells were harvested and washed with phosphate buffer saline
(PBS), and total RNA was extracted from the cells using Trizol reagent (Gibco
BRL) according to the manufacturer’s protocol. 3 mg of total RNA was used for
reverse transcription in a total volume of 20 ml with the Superscript
preamplification system (Promega, Madison, USA). Aliquots of 1.5 ml cDNA were
subsequently amplified in a total volume of 20 ml using the Gene amp PCR
kit following conditions recommended by the manufacturer. The sense and
antisense primers for Bcl-XL were 5‘-TTGGACAATGGACTGGTTGA-3‘
and 5‘-GTAGAGTGGATGGTCAGTG-3‘ (780 bp) respectively. The sense
and antisense primers for the b-actin gene used as an internal control were 5‘-GGTGGCACCTGTGGTCCACCT-3‘
and 5‘-CTTCACTTGTGGCCCAGATAG-3‘ (420 bp), respectively. The
cycling conditions were 94 ºC for 4 min, followed by 30 cycles at 94 ºC for 30
s, at 60 ºC for 30 s, and at 72 ºC for 1 min and a final extension at 72 ºC
for 10 min. PCR products were separated on the 1.5% agarose gel stained with
ethidium bromide and viewed under ultraviolet light.
Immunofluorescence microscopy
Transfected and untreated cells were seeded and grown on cover slips
in 6-well plates. After 24 h they were washed twice with PBS and fixed with
methanol acetic acid (3:1) for 15 min at room temperature. The cells were permeabilized
with PBS containing 0.25% Triton X-100 (Amresco) and 5% dimethyl sulfoxide
(DMSO) (Sangon, Shanghai, China) for 30 min at 37 ºC and washed twice with PBS
containing 0.25% Triton X-100. Cells were then incubated with the Bcl-XL
primary antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) for 60 min at 37
ºC. The anti-Bcl-XL was used at the dilution of 1:100 in PBS. After washing for
three times, the cells were incubated with the rabbit anti-mouse RPE-conjugated
secondary antibodies (BD PharMingen, San Diego, USA) for 60 min at 37 ºC and
washed three times with PBS. The cover slips were directly observed under
fluorescence microscope (Olympus Company, Ishikawa-cho, Hachioji-shi, Tokyo,
Japan), and the data were acquired with Pixera Camera (Pixera Corporation, Los
Gatos, USA).
Apoptosis analysis
Cell apoptosis was identified by fluorescence staining with acridine
orange (AO; Sigma, St. Louis, USA). Cells were collected from the above group
and washed once, resuspended in PBS, then 25 ml of the cell suspension
was mixed with 1 ml of a dye mixture containing AO (100 mg/ml) in PBS. One drop of
the stained cell suspension was placed on a microscope slide and observed under
fluorescence microscope.
Apoptosis was also determined by flow cytometry analysis. Cells were
washed twice with 0.01 M PBS and fixed with 70% ethanol. The cells were then
washed once with PBS, digested by 200 ml RNase (1 mg/ml) at 37 ºC for 30 min, and
stained with 800 ml propidium iodide (50 mg/ml) at room temperature for 30 min. Cells
were subject to flow cytometry analysis (EPICS-XL, Beckman Coulter, Fullerton,
USA) and data were analyzed with Multipcycle software.
Statistical analysis
Statistical analysis was performed using SPSS software (Release
11.0, SPSS Inc., Chicago, USA). Data were expressed as mean±SD
and analyzed by one-way analysis of variance (ANOVA) and least significant
difference (LSD) test; and P<0.05 was considered significant.
Results
siRNA synthesized from DNA templates efficiently inhibited the
transfected GFP gene and endogenous Bcl-XL gene in mammalian
cells
Extracted plasmids were primarily confirmed by agarose gel
electrophoresis [Fig. 1(A)]. We then used sequencing to verify the GFP
or Bcl-XL siRNA inserted templates [Fig. 1(B)].
First, the GFP siRNA or negative siRNA together with GFP expression
vector were transfected into MGC-803 cells. After 48 h, cells were collected
and washed twice with PBS, and directly observed under a fluorescence
microscope. No significant changes of GFP expression were found in cells
transfected with negative siRNA vector [Fig. 2(A), 1]. In contrast, the
GFP siRNA vector greatly diminished its expression [Fig. 2(A), 2]. The
lack of complete inhibition may be due, in part, to the high level of GFP
expression that was directed by a strong CMV promoter.
Next, we used vector-expressing siRNA to repress the endogenous Bcl-XL
gene. MGC-803 cells were transfected with either the negative vector or Bcl-XL
siRNA vector which directs synthesis of Bcl-XL siRNA. After 48 h, protein
expression was observed under a fluorescence microscope. Both the untreated
control group and the negative siRNA group showed similar fluorescence
expression [Fig. 2(B), 1 and 3]. But Bcl-XL expression in cells
transfected with Bcl-XL siRNA vector was reduced to the control level with the
secondary antibody alone [Fig. 2(B), 2 and 4]. Consistent with this, the
RT-PCR results showed that siRNA greatly reduced the mRNA of Bcl-XL (Fig.
3).
Down-regulation of Bcl-XL increases the spontaneous apoptosis of
cells
Having demonstrated that Bcl-XL siRNA can down-regulate the expression
of Bcl-XL, we asked whether there is higher spontaneous apoptosis in cells
transfected with Bcl-XL siRNA. First, we examined the morphological changes
in the cells. The nuclei of Bcl-XL siRNA transfectants exhibited bright condensed
chromatin or were fragmented, and some cells were blebbed. In contrast, the
untreated cells and negative siRNA transfectants did not show these apoptotic
features [Fig. 4(A)]. We then analyzed the Bcl-XL siRNA cell cycle
by flow cytometry. The Bcl-XL siRNA group had a higher proportion of cells
in the sub-G1 population than the negative siRNA group or the untreated group, 21.17%±1.26%
vs. 1.19%±0.18% and 1.56%±0.15% respectively, P<0.05
vs. negative siRNA or untreated control [Fig. 4(B)].
Discussion
The results presented in this study show that RNAi is effective in
treating the human gastric cancer cell line MGC-803 and that this phenomenon has
potential applicability as a therapeutic approach to gastric cancer treatment.
So far, there are several methods to produce siRNA, including chemical
synthesis, in vitro transcriptional synthesis, and vector-expressing
siRNA [17–19]. Here, we used the
vector-expressing hairpin siRNA method. First, we transiently co-transfected
GFP siRNA vector and GFP vector, observed the down-regulation of the exogenous GFP
gene expression, and demonstrated that it is feasible to use the
vector-expressing siRNA method in MGC-803 cells. Then we stably transfected
the Bcl-XL siRNA vector to MGC-803 cells.
Cancer chemotherapy mitigate the adverse side effects of drugs by
molecular targeting [20]. It has been reported that Bcl-XL is an anti-apoptotic
factor, in fact, cells over-expressing Bcl-XL showed resistance against a
variety of cellular stress [21–23], so
we chose Bcl-XL as the target gene to observe whether Bcl-XL siRNA could
increase spontaneous apoptosis. From morphological study to cell cycle
analysis, our results demonstrated that Bcl-XL siRNA indeed increased
spontaneous cellular apoptosis.
In conclusion, the study attempted to explore the functionality of
the RNAi pathway in gastric cancer cell lines MGC-803 and to evaluate the
biological impact of the phenomenon. Further research is necessary to improve
the efficacy of the vector expressing siRNA delivery and action, to
investigate the drug sensitivity of cells by Bcl-XL siRNA combined with
chemotherapeutic drugs, and to determine the optimal therapeutic action by
combined RNAi for several different anti-apoptotic genes.
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