expression by RNA interference suppresses human lung cancer
Yun-Chao Huang2*, Qi Zhang1, Zhi-Peng Hong1, Yong-Chun
Zhou2, and Wan-Ling Chen3
Affiliated Hospital, KunMing Medical College, Yunnan,
Affiliated Hospital, KunMing Medical College,
Provincial Corps Hospital, Chinese People's Armed Police
Accepted 22 July, 2011
Aquaporins (AQPs) represent a family of homologous water
channels expressed in many epithelial and endothelial cells.
Most tumors have been shown to exhibit high vascular
permeability and interstitial fluid pressure. Here, we
tested the regulation on the expression of AQP3 by RNA
interference (RNAi) in the human lung cancer cell line (XWLC-05)
and observed the changes of proliferation and adhesion
ability. The results show that AQP3 mRNA transcripts were
decreased to 65 and 79%, respectively (P<0.05). Western blot
analyses also revealed the AQP3 protein was decreased to 53
and 73% (P<0.05). MTS assay showed that proliferation of
XWLC-05 was significantly inhibited by RNAi after 48 and 72
h transfection. The invasion assay demonstrated that the
number of XWLC-05 cells penetrating the membrane in the
transfected group was considerably lower than those in the
untransfected and negative control group (P<0.05). These
results indicate that vector-based shRNA could be used as a
potential tool to inhibit the expression of AQP3.
Lung cancer, RNA interference, AQP5, cell adhesion ability,
Lung cancer is a disease of uncontrolled cell growth in tissues of the lung. This growth may lead to metastasis, which means the invasion of adjacent tissues and infiltration beyond the lung. The vast majority of primary lung cancers are carcinomas of the lung, derived from epithelial cells. Lung cancer, the most common cause of cancer-related death in men and women, is responsible for 1.3 million deaths worldwide annually, as of 2004. Current therapies for lung cancer mainly take forms of surgery, radiotherapy and pharmacological approaches. Although, they are helpful to some degree none of them can offer a permanent cure. For patients with leading lung cancer, even extensive surgical operations combined with chemotherapy have not sufficiently brought about improved prognosis (Dovedi and Davies, 2009; Felip et al., 2010; Germain et al., 2010). In recent years, it has been demonstrated that gene therapy is one of the possible candidates for an innovative therapeutic approach for the treatment of advanced gastric carcinoma (Yamamoto et al., 2009; Li et al., 2010). Aquaporins (AQPs) are proteins embedded in the cell membrane that regulate the flow of water. They are "the plumbing system for cells" (Yan et al., 2010). Aquaporin genes can function in forming tetramers in the cell membrane to facilitate the transport of water and in some cases, other small solutes across the membrane. Their defects can lead to several human diseases (Agre and Kozono, 2003; Agre, 2006).
Human AQP1 is naturally expressed in erythrocytes and many epithelial and endothelial tissues, including kidney, choroids plexus, bile duct, gall bladder, eye lens,brain, and placenta (Cheng et al., 1997; Schrier, 2007). AQP3 acts as the membrane channel of water and other small solutes, which plays a major role in fluid homeostasis. AQP3 facilitates water transport in epidermal cell migration and glycerol transport in epidermal cell proliferation (Heymann et al., 1998). AQP3 plays an important role in the maintenance of water homeostasis. Inhibition of the AQP3 water channel can increase the sensitivity of prostate cancer cells to cryotherapy (Hara-Chikuma and Verkman, 2008). In addition, AQP3 is widely expressed in the normal respiratory tract. Lung carcinomas, especially adeno-carcinomas, can produce AQP3, possibly in connection with their functional and/or biological nature, although, the detailed mechanism of AQP3 expression in lung carcinomas remains to be clarified (Liu et al., 2007).
RNA interference (RNAi) is an evolutionarily conserved process in which recognition of double-stranded RNA (dsRNA) ultimately leads to posttranscriptional suppression or gene expression. This suppression is mediated by dsRNA (21 to 23 nucleotides), which induces degradation of mRNA based on complementary base pairing (Elbashir et al., 2001; Brummelkamp et al., 2002).
In this study, we attempted to observe the down-regulation of AQP3 gene induced by vector-based small hairpin RNA (shRNA) in human lung cancer cells, and detected the changes of proliferation and adhesion ability after AQP3 gene was suppressed.
Cells and culture conditions
The human lung cancer cell lines
XWLC-05 were supplied by the
Institute of Clinical Cancer,
Kunming Medical College, China.
These cell lines were cultured
in RPMI 1640 (Sigma, St. Louis,
MO, USA) at 37°C in an
atmosphere of 5% CO2
in the air with 10% fetal bovine
Carlsbad, CA, USA).
Construction of pAQP3-siRNA
DNA sequence of AQP3 (GenBank
no: BC013566) was inputed into
Dharmacon web-based software for
selecting target sequences. The
following criteria were used to
identify targets for siRNAs from
the AQP3 cDNA coding sequence:
(a) start with an AA
dinucleotide; (b) 21 nucleotides
in length; (c) G/C content of <
50% and (d) no sequence homology
to other coding sequences on
BLAST search. Using these
criteria, two siRNA sequences
from human AQP3 were chosen as
5′- GAGAAUGUGAAGCUGGCCC-3′ (A2).
One negative control siRNA
containing a scrambled sequence
with the same nucleotide
composition was also selected.
Sense and antisense primers
containing the sense siRNA
sequence, 9 bp loop sequence,
antisense siRNA sequence, and
RNA polymerase III terminator
sequence were created at BamH
I and Hind III
restriction sites on the 5′ and
3′ ends, respectively. These
primers were annealed and
inserted into pSilencer 4.1-CMV
neo (Ambion, Inc., Austin, TX)
downstream of the H1RNA
polymerase III promoter
following the manufacturer’s
instructions. The resulted
plasmids containing siRNA
sequences A1, A2 and negative
control sequence NC were named
pAQP3-siRNA1, pAQP3-siRNA2 and
Cells were cultured in the RPMI
1640 medium, seeded at 1×106
/well in 6 well plates.
When the cells reached 80 to 90%
confluence, transfection of
dsRNA was performed with
Lipofectamine 2000 (Invitrogen,
USA) according to the
Briefly, one day before
transfection, the attached cells
in logarithmic growth phase were
respectively implated into
12-well plates at density of
Transfection mixes containing
Solution A (5
plasmid + 100 μl OptiMEM medium)
and Solution B (10 µl
+ 100 μl OptiMEM medium) were
prepared in 96-well plates. When
cells reached 70% confluence,
the plasmid DNA was transfected
into the cells. The transfection
efficiency was observed by
fluorescence microscopy after 24
h. Cells were transfected for 6
h per day, lasted for 3 days and
were collected in the 4th day.
The transfection concentration
of the experimental group shRNA
was 1.0, 2.5 and 5.0 μg/L
RNA isolation and RT-PCR
Total RNA was extracted with
Trizol reagent according to the
protocol described by the
supplier (TakaRa, Dalian,
China). cDNA was obtained from
the total RNA by reverse
transcriptase polymerase chain
reaction (RT-PCR) according to
the standard protocols (Wang et
al., 2009). PCR primers were
designed as follows: (300bp)
Amplification reactions (25 μl)
contained 1μl (50 ng) of cDNA, 2
μl (10 μmol/L) of each primer,
0.5 μl (10 μmol/L) of probe,
12.5 μl of PCR mix and 7 μl of
deionized water. Mixed them
gently and amplified by real
time RT-PCR, cycling parameters
were 55°C for 2 min, then 35
cycles of 93°C for 10 min, 93°C
for 10 min and 60°C for 55 min.
β-actin was used as
control. The targeted DNA
amplified specifically was
confirmed by electrophoresis and
sequencing. PCR products were
analyzed using GelWorks
software, after the ethidium
bromide-stained 1.5% agarose gel
Cells transfected with various
vectors were lysed in RIPA
buffer (1% NP-40, 50 mmol/L Tris,
150 mmol/L NaCl) 48 h after
transfection. Proteins in an
equal amount were separated by
gel electrophoresis and
a nitrocellulose membrane. The
membrane was blocked with 5%
nonfat milk and then incubated
with 1:1000 dilution of primary
antibody overnight at 4°C.
Subsequently, the membrane was
washed and incubated with a
antibody for 1 h after being
washed. Antibody binding was
detected by using an enhanced
system. Western immunoblotting
films were digitized, and band
intensities were quantified by a
Millipore Digital Bioimaging
System (Bedford, MA). β-actin
protein ratio was calculated for
Cell proliferation was assayed
by the CellTiter 96 AQ
proliferation assay (MTS) from
Promega (Madison, WI). After 24,
48 and 72 h, 20 μl of freshly
prepared MTS/PMS solution was
added to each well, and the
mixture was incubated for 2 h at
37°C. Optical density was read
directly at 490 nm using the
ELISA plate reader. All samples
were assayed at least in
quadruplicate with an
Matrigel invasion assay
Matrigel matrix (Becton
Dickinson Labware, Franklin
Lakes, NJ) was applied and
polymerized in 24-well 9-mm
inserts containing polyethylene
terephtphalate (PET) membranes
with 8-µm pores to create
invasion chambers as directed by
the supplier (Becton Dickinson).
Matrigel was thawed out at 4°C
overnight on ice. Pipettes,
plates and tips were chilled at
-20°C. Matrigel (5 mg/ml) in
serum-free cold DMEM was diluted
with pre-cooled pipettes. 100 μl
(h = 100 ul/200 mm 2 = 0.5 mm)
of the diluted matrigel per well
may be gently pipetted using a
pre-cooled pipette to ensure
homogeneity. The transwell was
incubated at 37°C for 30 min to
5 h for gelling.
Cells were harvested when they
grew to near confluence by
trypsinization, which was
inactivated with medium
containing bovine calf serum,
and cells were subsequently
washed twice in DMEM without
added serum or proteinase
inhibitor. 100 μl of the cell
suspension was put onto the
matrigel. Lower chamber of the
transwell was filled with 600 μl
(3 mm) of culture medium
containing 5 μg/ml fibronectin,
as an adhesive substrate.
Incubated at 37°C for 48 h, then
removed transwells from 24-well
plates and stained with
Diff-Quick solution. Noninvaded
cells were scraped off on the
top of the transwell with a
cotton swab, and invaded cells
were counted on the transwell
under a light microscope.
Experiments were performed at
least in duplicate, and a
typical set of data was
indicated. Differences were
statistically evaluated by
Student’s t-test. A P-value of
less than 0.05 was considered to
be statistically significant.
Inhibition of AQP3 expression by pAQP3-siRNA
To determine the inhibitory effect of shRNA on AQP3, three groups were divided: (1) non-transfected control group, (2) pAQP3-siRNA1 transfected group and (3) pAQP3-siRNA2 transfected group. AQP3 mRNA levels were assessed by RT- PCR, using β-actin as the internal control. 2 days after transfection, AQP3 mRNA was reduced by 35 and 21% in pAQP3-siRNA1 and pAQP3-siRNA2 groups, respectively compared to the control group (P<0.05), and the inhibition level of pAQP3-siRNA1 was significantly better than that of pAQP3-siRNA2 (Figure 1). Western blot analysis showed the suppression effect on AQP3 protein appeared at 48 h post-transfection. Total proteins from the non-transfected control group, pAQP3-siRNA1 and pAQP3-siRNA2 transfected groups were extracted 2 days after transfection and Western blot analyses were performed (Figure 2). Expression of AQP3 decreased significantly in the presence of XWLC-05 shRNA. The inhibitory rates were 47 and 27% by pAQP3-siRNA1 and pAQP3-siRNA2, respectively (P<0.05, compared to the control group). The results show that the inhibition level of pAQP3-siRNA1 was significantly better than that of pAQP3-siRNA2, which was similar to the result of mRNA analysis.
In this study, the effect of AQP3-shRNA on the growth of XWLC-05 at 24, 48 and 72 h after transfection were investigated (Figure 3). The results show that cell proliferation was not significantly inhibited at 24 h after
transfection. However, at 48, and 72 h post-transfection, the cell proliferation was significantly inhibited (P<0.05), suggesting silencing of AQP3 could inhibit the growth of lung cancer XWLC-05.
Matrigel invasion assays
The lung cancer cell invasion of Matrigel matrix was inhibited when the human lung cancer XWLC-05 cell was transfected by the pAQP3-siRNA1 (Figure 4). It could effectively decrease invasion at 24, 48 and 72 h after tranfection (P<0.05 as compared to control). In addition, there was a statistically significant difference between 24 h group, and the post-transfection 48 and 72 h groups (P<0.05). However, such significant difference did not appear between the 48 and 72 h transfected groups (P>0.05).
Aquaporins are membrane water channels that play critical roles in controlling the water contents of cells. Aquaporins have been proposed as a novel target in cancer and oedema, and are associated with surprising arrays of important processes in the brain and body, such as angiogenesis, cell migration, development and neuropathological diseases. Based on the fact that current therapies for both cancer and brain are limited, new pharmacological approaches focusing on AQPs may offer exciting potentials for clinical advance (Yool et al., 2010). AQP1-null mice remarkably impaired tumor growth after subcutaneous or intracranial tumor cell implantation, with reduced tumor vascularity and extensive necrosis (Saadoun et al., 2005). AQP1 expression was intensely up-regulated in all glioblastomas studied (Oshio et al., 2005). AQP3 acts as the membrane channel of water and other small solutes and plays a major role in fluid home-ostasis. It can play an important role in the maintenance of water homeostasis. AQP3 is widely expressed in the normal respiratory tract. Lung carcinomas, especially adenocarcinomas, can produce AQP3, possibly in connection with their functional and/or biological nature (Liu et al., 2007).
Moreover, some reports showed that down-regulating the expression of AQP5 using siRNA approaches may inhibit the proliferation and invasion of cancer cells (Chen et al., 2006; Frigeri et al., 2007). RNA interference has become widely used in vivo knockdown of genes in cancer therapy. This study aimed to determine whether the silencing of AQP3 RNA interference may inhibit the growth of human lung cancer cell. The AQP3 gene expression was markedly decreased by RNAi based on RT-PCR and Western blot results, the proliferation of XWLC-05 was significantly inhibited at 48 and 72 h post-transfection, and the adhesion ability of XWLC-05 cells was also significantly decreased compared with the untransfected group and negative control group (P<0.05). The invasion assays demonstrated that the number of XWLC-05 cells penetrating the membrane in the transfected group was significantly lower than those in the untransfected and negative control groups (P<0.05). In summary, AQP3 gene expression silenced by shRNA could inhibit the growth and invasion ability of lung cancer cells.
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