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 Pdf file on shRNA targeting HDGF suppressed cell growth
and invasion of squamous cell lung cancer
Jie Meng1,2,
Wei Xie3*, Liming Cao1, Chengping Hu1, and
Zhiyuan Zhen1
1Department of
Respiratory Medicine, 2Department of
Pharmacology, 3Department of
Cardiovascular Medicine,
*Correspondence
address. Tel/Fax: +86-731-84327105;
E-mail: [email protected]
Hepatoma-derived
growth factor (HDGF), a nuclear protein with both mitogenic and angiogenic
activity, has been reported to be mainly involved in tumorigenesis and the
progression of non-small cell lung cancer. In this study, the HDGF expression was
knocked down by specific-shRNA with lentivirus expression vector targeting HDGF in lung squamous
cell carcinoma 520 cells. HDGF knocked down by shRNA suppressed the cell
proliferation significantly both in vitro and in vivo as indicated by MTT, plate clone and transplanted tumor
model assays. In addition, the knocked-down expression of HDGF also inhibited
cell migration and invasion as shown in transwell and Boyden experiments. We
concluded that HDGF acts as an oncogene participating in the pathogenesis of
squamous cell lung cancer, and HDGF may be a key therapeutic target for
non-small cell lung cancer.
Keywords hepatoma-derived growth factor; squamous cell lung cancer; oncogene
Received: August 30,
2009 Accepted: October 22, 2009
Introduction
Lung cancer is one of
the main causes of cancer death worldwide. The disease is more common in
countries with high tobacco consumption, including Hepatoma-derived
growth factor (HDGF), a heparinbinding growth factor originally purified from
media conditioned with the human hepatoma cell line HuH-7, can stimulate
proliferation of NIH cells [2]. HDGF has a mitogenic function for various types
of cells such as human hepatocellular carcinoma cells, fibroblasts, endothelial
cells, vascular smooth muscle cells, and fetal hepatocytes. HDGF can
translocate to the nucleus by nuclear localization signals, and its nuclear
translocation is essential for the induction of cell growth activity.
Previous studies
manifested that overexpressed HDGF is a metastatic and prognostic factor for
lung cancer [3–5]. Downregulation of
HDGF expression by chemically synthesized shRNA could suppress cell growth and
invasion in vitro [6]. Previous results have indicated that HDGF is a unique nuclear/growth
factor that plays an important role in the development and progression of lung
cancer. In this study, the expression level of HDGF and its roles in cells were
identified in squamous cell carcinoma 520 cell line.
Materials and Methods
Cell lines and
animals
The human lung
squamous cell carcinoma 520 cell line was grown in RPMI 1640 medium (Hyclone,
Logan, USA) supplemented with 10% new born calf serum (PAA Laboratories Inc.,
Queensland, Australian). BALB/c nude mice, 4–6 weeks old, 18Detection of HDGF mRNA expression
in lung squamous cell carcinoma 520 cells by RT-PCR Expression of HDGF mRNA in lung squamous cell carcinoma 520 cell
lines was detected. Total RNA was prepared by using Trizol reagent (Takara,
RNAi sequence
design against HDGF and construction of vectors expressing HDGF shRNA
HDGF-specific target
sequence was chosen according to online shRNA tools of Invitrogen
(http://www.invitrogen. com/rnai) using the HDGF reference sequence (GenBank accession No.
NM_004494.2). The target sequence was designed as follows: top strand, HDGF 5‘-CACCGCCGGCAGAAGGAGTACAAAAACGTTTGTACTCCTTCTGCCGG-3‘; bottom strand, 5‘-AAAACCGGCAGAAGGAGTACAAACGTTTTTGTACTCCTTCTGCCGGC-3‘. Then shRNAs were
chemically synthesized and lentiviral vector was constructed as described
previously [7]. The exact insertion of the specific shRNA was further confirmed
by sequencing.
Plasmids and
transfection
shRNA-HDGF and control
plasmids were transfected into lung squamous cell carcinoma 520 cells by
Lipofectamine 2000. To generate stable clones, transfected cells were selected
with 5 mg/ml blasticidin. The medium was changed every 3–4 days until blasticidin-resistant colonies appeared. Single colonies were
picked and grown in selection medium.
Detection of HDGF mRNA expression
in cells by real-time qPCR
Total RNA of these
cell clones was prepared and real-time qPCR was performed to examine the HDGF mRNA expression. GAPDH gene was used as a
normalizing control. The designed paired primers were as follows: HDGF, 5‘-CAGCCAACAAATACCAAGTCT-3‘ (forward), 5‘-GTTCTCGATCTCCCACAGC-3‘ (reverse);
GAPDH, 5‘-GAAGGTCGGAGTCAACGG-3‘ (forward), 5‘-TGGAAGATGGTGATGGGATT-3‘ (reverse).
The PCR reaction was carried out in a volume of 20 ml using SYBR green mix
(Takara) on MXP3000 instrument (Stratagene Laboratories,
Western blot
analysis of HDGF protein
Western blot was
performed as described previously [7]. In brief, cells were washed twice with
cold phosphatebuffered saline (PBS) and lysed on ice in RIPA buffer with
protease inhibitors (Shanghai Biocolor BioScience & Technolgy Company,
MTT assay
The cells were seeded
into 96-well microtiter plates at a density of 1000 cells per well. The cells
were incubated for 1, 2, 3, 4, 5, 6, and 7 days, and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was
performed by adding 20 ml of 5 mg/ml MTT (Promega, Madison, USA) for 4 h. When MTT incubation was
completed, supernatants were removed. Then 150 ml of dimethyl
sulfoxide (Sigma,
Soft agar clone
formation
Cells were planted in
24-well plastic plates spread with low-melting point agar (0.35% in the upper
layer and 0.6% in the low layer). Each type of cell was planted in five wells,
with 100 cells in each well. The cells were cultured at 37ºC, with 5% CO2
and under saturation humidity for 14 days. Cells clones with more than 50 cells
were counted under an invert microscope. Clone form rate was calculated as
follows.
EQ
In vitro migration and
invasion assay
Total of 1 ´105 cells
were seeded on a fibronectin-coated polycarbonate membrane insert with a pore
size of
Tumor
transplantation in nude mice
The 1 ´106
logarithmically growing cells were injected subcutaneously into 4–6 weeks old male BALB/c nude/ nude mice. Each experimental group consisted
of three mice. After 4 weeks of observation, the mice were sacrificed and
tumors were stripped. Tumor weight was measured and tumor volume was calculated
according to the formula
EQ
Finally, the HDGF
expression was examined again by real-time PCR in implanted nude mice of
shRNA-HDGF and control cell groups.
Statistic
analysis
SPSS13.0 package
(Abbott Laboratories,
Results
HDGF mRNA is highly
expressed in lung squamous cell carcinoma 520 cells and shRNA sequence is
confirmed by sequencing
HDGF is highly expressed in
lung squamous cell carcinoma 520 cells, and its expression density is nearly
equal to that of housekeeping gene GAPDH [Fig. 1(A)]. shRNA sequence inserted into lentivirus expression vector was
identified, which was consistent with the designed sequence [Fig. 1(B)].
ShRNA targeting HDGF suppressed the
expression of HDGF at mRNA and protein levels in lung squamous cell carcinoma
520 cells
shRNA targeting HDGF was used to decrease
the expression of HDGF mRNA in lung squamous cell carcinoma 520 cells. Ten single cell clones
with the integration of shRNA-HDGF lentivirus vector was selected to examine
the silencing efficiency compared with control 520 cells by real-time
quantitative PCR, and cell clones shRNA-HDGF-2 and -5 revealed decreased HDGF mRNA by up to 73.1%
and 76.7% respectively [Fig. 1(C)]. To further confirm the specificity of shRNA-mediated silencing of HDGF,
the expression of HDGF protein was also determined by western blot. As shown in
Fig. 1(D), HDGF protein of both
cell clones, shRNA-HDGF-2 and -5, was decreased by 71% and 73%, respectively,
compared with the control shRNA-Ctr 520 cells. shRNA-HDGF-5 cell clone was used
in subsequent experiments as an HDGF knockdown cell model.
Reduction of HDGF
expression inhibited cell growth
The growth curves
determined by MTT assays showed significant proliferation suppression in
shRNA-HDGF cells compared with control shRNA-Ctr 520 cells [Fig. 2(A)]. shRNA-HDGF cells
yielded significantly fewer colonies than control cells (P< 0.01) according to soft agar clone formation assay (n = 3) (Table 1). Anchorage-dependent growth in cells treated with shRNA-HDGF
transfection was also reduced compared with control cells. Weight and volume of
tumors were dramatically lighter and smaller (P< 0.05) in shRNA-HDGF cells-implanted nude mice than those implanted with
control cells (n =
3) [Fig. 2(B)]. Real-time
quantitative PCR showed that HDGF expression was obviously reduced in implanted
nude mice of shRNA-HDGF cell groups compared with control cell groups [Fig. 2(C)].
Reduction of HDGF
expression suppressed cell migration and invasion in vitro
In vitro studies were performed
to detect the effects of HDGF repression on both migration and invasion in 520
cells. As shown in Fig. 2(D), inhibition of HDGF expression resulted in decreased migration capacity
of 520 cells (P<
0.05). As compared
with control cells, the shRNA-HDGF cells showed a significant decrease in
invasion capacity (P<
0.05) [Fig. 2(E)].
Discussion
In this study, we determined
the expression of HDGF in 520 cell line, and constructed lentivirus vector that
could stably repress HDGF expression in these cells. We observed that the
decreased expression of HDGF by sequence specific shRNA can inhibit the growth
of 520 cells both in vivo and in vitro. In addition, we observed that inhibition of HDGF expression by shRNA
suppressed the migration and invasion capacity of 520 cells. HDGF encodes a member of
the hepatoma-derived growth factor family. The encoded protein has a wide range
of biological functions in cellular biology, such as mitogenic activity [8],
promoting angiogenesis [9] and lung remodeling [10]. It is reported that HDGF
is overexpressed in lung cancer tissues [3–5]. In agreement with these studies, we also observed high level of HDGF expression in 520 cell
line. This indicated that HDGF might play a role in the development of lung
cancer.
Clinical studies have
shown that overexpression of HDGF is associated with poor outcome of tumors
such as gastric [11,12], lung cancer [3–5], liver cancer [13], colorectal stromal tumors [14], and pancreatic
cancer [15] in human. It is also reported that HDGF transfection significantly
activated ERK1/However, it remains
unclear the mechanisms of HDGF contributing to the proliferation and invasive
capacity of 520 cells. There are reports showing that HDGF knockdown can lead
to the induction of the expression of the pro-apoptotic protein Bad and inactivation
ERK and Akt, the latter in turn led to dephosphorylation of Bad and activation
of the intrinsic apoptotic pathway [17]. It is deserved to determine whether
HDGF contributes to the development of lung cancer in similar pathways in
future. In conclusion, HDGF may play a key role in promoting the growth and
invasion of lung squamous cell carcinoma 520 cells. Lentivirus-mediated shRNA
vector can stably down-regulate HDGF expression and inhibit the proliferation
and invasion capacity of 520 cells. HDGF may serve as a potential target for
the treatment of lung cancer in patients with HDGF overexpression.
Funding
This work was
supported by a grant from the Natural Science Foundation of Hunan Province (No.
07FJ4159).
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