Insulin-Like Growth Factor 1 Receptor (IGF-1R) as a Target of MiR-497 and Plasma IGF-1R Levels Associated with TNM Stage of Pancreatic Cancer
et al. (2014) Insulin-Like Growth Factor 1 Receptor (IGF-1R) as a Target of MiR-497 and Plasma IGF-1R Levels
Associated with TNM Stage of Pancreatic Cancer. PLoS ONE 9(3): e92847. doi:10.1371/journal.pone.0092847
Insulin-Like Growth Factor 1 Receptor (IGF-1R) as a Target of MiR-497 and Plasma IGF-1R Levels Associated with TNM Stage of Pancreatic Cancer
Jian-Wei Xu 0
Tian-Xiao Wang 0
Lei You 0
Lian-Fang Zheng 0
Hong Shu 0
Tai-Ping Zhang 0
Yu-Pei Zhao 0
Lucia R. Languino, Thomas Jefferson University, United States of America
0 1 Department of General Surgery, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China , 2 Department of Head and Neck Surgery, Beijing Cancer Hospital and Institute, Peking University Cancer Hospital , Beijing , China , 3 Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College , Beijing , China
The expression levels and regulatory roles of miR-497 in pancreatic cancer are unclear. The clinical value of plasma insulinlike growth factor 1 receptor (IGF-1R) in pancreatic cancers has not been investigated. In the present study, we demonstrated that miR-497 was significantly downregulated in pancreatic cancer tissues. Upregulation of miR-497 in BxPC-3 and AsPC-1 pancreatic cancer cell lines inhibited proliferation, enhanced apoptosis, re-sensitized cells to gemcitabine and suppressed IGF-1R and p-AKT expression through direct downregulation of IGF-1R protein expression. Opposite effects were observed after downregulation of miR-497. Plasma IGF-1R levels in patients with pancreatic cancer increased significantly, compared with that in patients with chronic pancreatitis, other pancreatic tumors and pancreatic neuroendocrine tumors (P = 0.006, P = 0.018 and P = 0.004, respectively), and displayed potential values for distinguishing pancreatic lesions. However, the levels in pancreatic cancer patients were comparable to that in healthy volunteers (P = 0.095). The tumor locations and TNM stage were associated with plasma IGF-1R levels (P = 0.013 and P = 0.01, respectively). There was no significant difference of overall survival between high and low IGF-1R expression groups. In conclusion, we demonstrated that miR-497 attenuated the malignancy of pancreatic cancer cells and promoted sensitivity of cells to gemcitabine by directly downregulation of IGF-1R expression. Plasma IGF-1R displayed a potential value for distinguishing pancreatic lesions and could be a new biomarker for guiding TNM stage of pancreatic cancer.
Funding: This study was supported by grants from the National Natural Science Foundation of China (No. 81272484, 81141027), Beijing Natural Science
Foundation (No. 7132179), Beijing Municipal Natural Science Foundation (7100003) and the Research Special Fund for Public Welfare Industry of Health
(201202007). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and
devastating disease. PDAC has an extremely poor prognosis with a
5-year survival rate lower than 5% . Although the mechanisms
of pancreatic carcinogenesis, the new markers for early detection,
and the new therapeutic strategies have been widely investigated
[2,3,4], the overall survival has not been improved in the past 80
years . The molecular mechanisms of the progression of PDAC,
including proliferation, apoptosis, drug resistance, remain unclear.
MiRNAs are short non-coding RNAs, which can inhibit
translation of messenger RNA (mRNA) into protein by binding
to the 39-untranslated region (39-UTR). MiRNAs can act as
oncogenes or tumor suppressors in the regulation of
carcinogenesis, metastatic capacity and drug resistance . Downregulation
of miR-497 has been observed in breast, colorectal and cervical
cancers [7,8,9]. However, there is no report about miR-497
expression levels in pancreatic cancer at present. Insulin-like
growth factor 1 receptor (IGF-1R) was identified as a target of
miR-497. Downregulation of miR-497 contributes to the
malignancy of colorectal cancer and cervical cancer by upregulating
IGF-1R [8,9]. However, the regulatory roles and mechanisms of
miR-497 in pancreatic cancer are still unclear.
IGF-1R is a tyrosine kinase receptor, which involves in the
regulation of proliferation, apoptosis, differentiation and
malignant transformation of cancer cells . Upregulation of IGF-1R
in human PDAC tissues has been reported  and associates
with higher tumor grade and poor survival . Nevertheless, the
plasma IGF-1R levels in pancreatic cancer patients were not
detected in previous studies. The clinical values of plasma IGF-1R
in pancreatic cancer are unknown.
In the present study, we found that miR-497 was significantly
downregulated in pancreatic cancer tissues. Upregulation of
miR497 inhibited proliferation, enhanced apoptosis and promoted
sensitivity to gemcitabine through directly downregulating IGF-1R
expression in PDAC cancer cells in vitro. We also showed that the
levels of plasma IGF-1R in pancreatic cancer patients were
comparable to that in healthy volunteers, but higher than that in
patients with chronic pancreatitis, other pancreatic tumors and
pancreatic neuroendocrine tumors, and displayed values for
distinguishing pancreatic lesions. The tumor locations and TNM
stage were associated with plasma IGF-1R levels. There was no
significant difference of overall survival time between high and low
IGF-1R expression groups.
Studies were performed in accordance with ethics approval
from the Institutional Review Boards of Peking Union Medical
College Hospital. Written informed consent was obtained from all
Detecting the expression of miR-497 by in situ
10 formalin-fixed, paraffin-embedded pancreatic cancer
specimens and matched tumor-adjacent tissues were obtained and
made into tissue microarrays. The expression levels of miR-497 in
tissues were detected using miRCURY LNA detection probe for
miR-497 (Exiqon, Vedbaek, Denmark, product number:
3825615). ISH was performed as following description. Briefly, slides
were incubated at 37uC for 30 min, deparaffinized in xylene, and
rehydrated with graded alcohol washes. Then slides were placed
into 4% paraformaldehyde for 20 min in a fume hood, and then
washed with phosphate buffered saline (PBS) three times. The
slides were then treated with 15 mg/ml proteinase K for 15 min at
room temperature. Thereafter, washed the slides with PBS and
fixed them in 4% paraformaldehyde for 15 min after that. After
rinsing, slides were pre-hybridized with hybridization buffer for 1
hour at room 50uC and then hybridized overnight at 4uC in the
hybridization buffer containing probe. Stringent washes were
carried out at 50uC for 20 min, and then the slides were incubated
in a blocking solution for 1 hour at room temperature.
Subsequently, slides were incubated in blocking solution with
alkaline phosphatase conjugated anti-DIG Fab fragment overnight
at 4uC. The colorimetric detection reaction was performed using
NBT/ BCIP kit (ThermoFisher Scientific) according to the
manufacturers protocol. The ISH results were recorded as the
percentage of positive cells.
Cell culture and reagents
BxPC-3 and AsPC-1 PDAC cell lines were kindly provided by
Professor Helmut Freiss (Heidelberg University, Germany), who
obtained from the American Tissue Type Culture Collection
(ATCC, Rockville, MD) [13,14,15]. PDAC cells were cultured in a
humidified incubator with 5% CO2 at 37uC in RPMI-1640
medium supplemented with 10% fetal bovine plasma (FBS,
Hyclone). The primary antibodies were purchased from Cell
Signaling Technology, including IGF-I Receptor b (D23H3) XP
Rabbit mAb ( #9750), b-actin (13E5) Rabbit mAb (#4970),
Phospho-Akt (Ser473) (D9E) XPRabbit mAb (#4060), Akt (pan)
(11E7) Rabbit mAb (#4685), Caspase-3 Antibody ( #9662) and
PARP Antibody ( #9542).
MiR-497 mimics (59- CAGCAGCACACUGUGGUUUGU-39,
59-AAACCACAGUGUGCUGCUGUU-39), mimics control
59-ACGUGACACGUUCGGAGAATT-39), miR-497 inhibitor
(59-ACAAACCACAGUGUGCUGCUG-39) and inhibitor control
(59-CAGUACUUFigure 1. The expression level of miR-497 in pancreatic tissues. MiR-497 levels in pancreatic tissues were detected using ISH (2006). (A)
Negative control. (B) ISH for miR-497 in pancreatic cancer tissue. (C) ISH for miR-497 in tumor-adjacent tissue. (D) The average percentage of positive
cells in pancreatic cancer tissues was 30.0%635.4%, which was significantly lower than that in tumor-adjacent tissues (93.5.0%62.4%). The data were
shown as mean6SD.
Figure 2. MiR-497 suppressed PDAC cells proliferation. Proliferation was analyzed by CCK-8 assay. (A, B) Transfection with miR-497 mimics
suppressed PDAC cells proliferation in AsPC-1 and BxPC-3 cells, respectively. (C, D) Transfection with miR-497 inhibitor promoted PDAC cells
proliferation. (* P,0.05).
UUGUGUAGUACAA-39) were synthesized by Genepharma
(Shanghai, China). MiRNAs at 50100 nM were transfected
using Lipofectamine 2000 transfection reagent (Invitrogen,
Carlsbad, CA) according to the manufacturers protocol.
Cellular RNA extraction and quantitative RT-PCR
Cells were transfected in 6-well plates. After 48 hours of
transfection, total RNA was extracted using TRIzol (Invitrogen,
Carlsbad, CA) according to the manufacturers protocol. For
IGF1R quantitative assay, total RNA was reverse transcribed using the
reverse transcription kit (Promega, Madison, WI) according to the
Figure 3. MiR-497 promoted chemosensitivity of gemcitabine. (A) AsPC-1 cells were transfected for 24 hours, and then treated with 100nM
gemcitabine for 48 hours. Upregulation of miR-497 by transfection of mimics re-sensitized cells to gemcitabine. Downregulation of miR-497 by
transfection of inhibitor decreased the sensitivity of cells to gemcitabine. (B) BxPC-3 cells were transfected for 24 hours, and treated with serially
diluted gemcitabine (1nM, 10nM, 30nM, 100nM). Cells transfected with miR-497 mimics were more sensitive to gemcitabine. (C) Cells transfected with
miR-497 inhibitor were more resistant to gemcitabine. (* P,0.05).
manufacturers instructions. Real-time PCR was performed using
the SYBR Green Master Mix (Takara, Japan). GAPDH were
served as the endogenous control.
IGF-1R Forward primer:
Reverse primer: 59-AACCATTGGCTGTGCAGTCA-39
GAPDH Forward primer:
For miR-497 quantitative assay, TaqMan miRNA assay were
used according to the manufacturers protocol (Applied
Biosystems). U6 was used as an endogenous control. Fold changes were
calculated using the 22DDCT method.
In vitro proliferation was analyzed using a cell count kit (CCK-8).
BxPC-3 cells and AsPC-1 cells were transfected in 6-well plates
(56105cells/well). After 24 hours, cells were trypsinized and
reseeded in 96-well plates (1000 cells/well). 10 ml/well CCK-8
reagent was added at 0, 24, 48, 72 hours, respectively, and
incubated for 2.5 hours at 37uC. Optical density (OD) was
measured at 450 nm and 630 nm by a microplate reader (Wellscan
MK3, Thermo/Labsystems, Finland).
BxPC-3 and AsPC-1 cells were transfected for 24 hours, plated
in 96-well plates (4000 cells/well), and treated with serially diluted
gemcitabine (Eli Lilly and Company) in triplicates. After 48 hours
incubation, 10 ml/well CCK-8 reagent was added and incubated
for 2.5 hours at 37uC. Optical density (OD) was measured at 450
nm and 630 nm using a microplate reader.
After 48 hours of transfection in 6-well plates, cells were
digested with trypsin solution and lysed with RIPA buffer
(Applygen, Beijing). Total proteins were separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to a polyvinylidene difluoride (PVDF) membrane
(Millipore, Billerica, MA). After blocking with 5% non-fat dry milk
at room temperature for 1 hour, the membranes were incubated
overnight at 4uC with primary antibodies. The membranes were
then washed and incubated with a horseradish
peroxidaseconjugated secondary antibody (Applygen, Beijing) at room
temperature for 1 hour. Protein bands were visualized with
echochemiluminescence (ECL) detection system, and the
expression levels of these proteins were evaluated using Image-Pro Plus
6.0 software (Media Cybernetics, USA).
Dual-luciferase reporter assay
The pmiR-RB-Report-IGF-1R-39-UTR vectors containing wild
type or mutated target sequence were constructed by RiboBio Co.,
Ltd. (Guangzhou, China). BxPC-3 cells were plated into 12 well
plates (16105 cells/well) and co-transfected with miR-497 mimics
and vectors expressing mutated target sequence, or co-transfected
with mimics and vectors expressing wild type target sequence
using lipofectamine 2000. After transfection for 48 hours,
luciferase activity was measured using the Dual-Luciferase Reporter
Assay System (Promega) according to the manufacturers protocol.
Patients and plasma IGF-1R expression
Plasma samples were collected from 42 pancreatic cancer
patients. Plasma samples from patients with other pancreatic
tumors (29 patients, including serous cystadenoma (7 cases),
mucinous cystadenoma (8 cases), solid pseudopapillary tumor (10
cases) and intra-ductal papillary mucinous neoplasm (4 cases)),
chronic pancreatitis (CP, 19 patients), pancreatic neuroendocrine
tumor (PNET, 19 patients) and healthy volunteers (30 cases) were
collected as controls. Pancreatic cancer, PNET and other
pancreatic tumors were diagnosed through pathological
examination. CP was diagnosed according to the clinical diagnostic
criteria. Blood samples were centrifuged at 3000 revolutions per
minute (rpm) for 10 minutes. Plasma was collected and stored at
80uC before use. The plasma IGF-1R levels were detected using
human IGF-1R ELISA Kit (Catalog number: CSB-E13766h,
CUSABIO, China) following the manufacturers protocol.
SPSS v.13.0 software (SPSS, Inc, Chicago, IL) was used for
statistical analyses. Continuous data were presented as mean 6
standard deviation (SD) and compared by analysis of variance
(ANOVA), students t test or the MannWhitney U test.
Categorical data were presented as percentage and compared
using a Pearson x2 test or Fisher exact test when cell counts were
,5. The Kaplan-Meier method was used for survival analysis
using the log-rank test. Statistical significance was defined as
P , 0.05.
MiR-497 was downregulated in pancreatic cancer tissues
MiR-497 levels in 10 pancreatic cancer specimens and matched
tumor-adjacent tissues were detected using ISH. The average
percentage of positive cells in pancreatic cancer tissues was
30.0%635.4%, which was significantly lower than that in
tumoradjacent tissues (93.5.0%62.4%) (P = 0.000).(Figure 1)
MiR-497 inhibited proliferation
After 48 hours of transfection with miR-497 mimics or
inhibitor, miR-497 levels were significantly upregulated or
downregulated (Figure S1 A, B). Furthermore, miR-497
upregulation significantly inhibited proliferation of PDAC cells
(Figure 2 A, B). In contrast, miR-497 downregulation promoted
the proliferation (Figure 2 C, D).
MiR-497 promoted sensitivity to gemcitabine
After gemcitabine treatment for 48 hours, the inhibition rates of
PDAC cells transfected with miR-497 mimics were significantly
higher than cells transfected with mimics control (Figure 3 A, B).
Cells transfected with miR-497 inhibitor were more resistant to
gemcitabine treatment than cells transfected with inhibitor control.
(Figure 3 A, C).
MiR-497 increased expression of cleaved caspase-3 and
PARP activated by gemcitabine
Caspase-3 and Poly (ADP-ribose) polymerase (PARP) are
essential for apoptosis. Transfected cells were treated with
gemcitabine for 48 hours, and then the levels of cleaved
caspase3 and PARP were detected by western blotting. We found that
upregulation of miR-497 increased the levels of cleaved caspase-3
and cleaved PARP (Figure 4 A, B, Figure S2). Conversely,
downregulation of miR-497 decreased the levels of cleaved
caspase-3 and cleaved PARP. (Figure 4 A, B, Figure S2).
MiR-497 suppressed IGF-1R protein expression by
binding to the 39-UTR
According to the prediction of open access databases
(TargetScan, miRBase Targets, PicTarget, microRNA.org), IGF-1R was
considered as a candidate target of miR-497. To confirm this
prediction, we performed a luciferase reporter assay. We found
Plasma expression of IGF-1R
luciferase activity was significantly decreased after co-transfection
of miR-497 mimics and vectors expressing wild type target
sequence in BxPC-3 cells, compared with that in cells
cotransfected with mimics and vectors expressing mutated target
sequence (P,0.05) (Figure 5A). These findings indicated that
IGF-1R was a direct target of miR-497.
To further confirm, western blotting was performed. We found
that upregulation of miR-497 suppressed the expression of
IGF1R protein without any change in the expression of IGF-1R
mRNA (Figure 5 B, C, D, Figure S3). In contrast,
downregulation of miR-497 increased IGF-1R protein expression (Figure
5 C, D, Figure S3). Additionally, we investigated the level of p
AKT mediated by IGF-1R. We observed that the level of p-AKT
decreased significantly after upregulation of miR-497. On the
contrary, inhibition of miR-497 increased the level of p-AKT
(P,0.05) (Figure 5 C, D, Figure S3).
The plasma IGF-1R levels in patients with pancreatic
Plasma IGF-1R levels were detected by ELISA. The plasma
levels of IGF-1R in patients with pancreatic cancer, chronic
pancreatitis, other pancreatic tumors, PNET and healthy
volunteers were 0.82360.57 ng/ml, 0.47260.42 ng/ml,
0.56260.3 ng/ml, 0.46060.21 ng/ml, 1.00460.50 ng/ml,
respectively. The levels in patients with pancreatic cancer increased
significantly, compared with that in patients with chronic
pancreatitis, other pancreatic tumors and PNET (P = 0.006,
P = 0.018, and P = 0.004, respectively.). There was no significant
difference of plasma IGF-1R levels between patients with
pancreatic cancer and healthy volunteers (P = 0.095). (Figure 6).
The diagnostic value of plasma IGF-1R
The diagnostic value of plasma IGF-1R was evaluated using
ROC curve. We showed that plasma IGF-1R displayed a value for
distinguishing pancreatic cancer from chronic pancreatitis and
PNET (AUC = 0.713, 95% CI: 0.5640.862, P = 0.008;
AUC = 0.711, 95% CI: 0.5810.840, P = 0.009, respectively).
When cut-off value defined at 0.257 ng/ml, the sensitivity and
specificity for distinguishing pancreatic cancer from chronic
pancreatitis were 95.2% and 47.3%. When cut-off value defined
at 0.699 ng/ml, the sensitivity and specificity for distinguishing
pancreatic cancer from PNET were 47.6% and 89.5%. Plasma
IGF-1R might also be used in differentiating pancreatic cancer
from other pancreatic tumors (AUC = 0.634, 95%CI: 0.504
0.763, P = 0.057). When cut-off value defined at 0.925 ng/ml, the
sensitivity and specificity for differentiating pancreatic cancer from
pancreatic benign tumors were 33.3% and 89.7%.
The correlation between plasma IGF-1R levels and
clinicopathological parameters and survival analysis
Patients were divided into high and low expression groups using
the 75th percentile of plasma IGF-1R levels. Tumor locations and
TNM stage were associated with plasma IGF-1R levels (P = 0.013,
P = 0.01, respectively. Table1). The median survival time was 18
months, and the 1-, 3-years survival rates were 64% and 23.1%,
respectively. The median survival times in high and low expression
groups were 11 and 18 months, respectively. No significant
difference of overall survival time between high and low expression
groups was observed (P = 0.366).
Decreased miR-497 expression has been demonstrated in
breast, colorectal and cervical cancers [7,8,9]. However, there is
no report about miR-497 expression levels in pancreatic cancer
tissues. Upregulation of miR-497 can suppress cancer cells
proliferation, promote apoptosis, decrease migration and invasion
capacities and increase chemosensitivity [16,17,9]. However, the
roles and mechanisms of miR-497 in pancreatic cancer remain
unknown. In the current study, we showed miR-497 was
significantly downregulated in pancreatic cancer tissues.
Upregulation of miR-497 inhibited cells proliferation, enhanced apoptosis
and promoted sensitivity to gemcitabine by directly
downregulating IGF-1R protein expression.
Our study identified the role of miR-497 in regulating the
malignancy of pancreatic cancer. The results supported that
miR497 inhibited cancer cells proliferation, promoted apoptosis and
increased chemosensitivity, as reported by literature [9,16,17].
Then, we investigated the mechanisms of miR-497 in regulating
the progression of pancreatic cancer. We found IGF-1R was a
direct target of miR-497 by a luciferase reporter assay, which was
also reported in colorectal cancers and cervical cancers [8,9]. We
also performed western blotting for further confirmation. We
showed that upregulation of miR-497 decreased IGF-1R protein
levels without any change in mRNA expression. Downregulation
of miR-497 increased IGF-1R protein levels. Additionally, we
investigated the expression of IGF-1R-mediated downstream
molecular. We found the level of p-AKT decreased significantly
after upregulation of miR-497. Inhibition of miR-497 increased
the expression of p-AKT. Therefore, we speculated that miR-497
attenuated the malignancy of pancreatic cancer by partly
suppressing IGF-1R/AKT pathway.
IGF-1R/AKT pathway has been identified to involve in the
regulation of multiple biological processes of cancers [18,19]. AKT
can activate BAD by phosphorylation on Ser136  or activate
NF-kB via regulating IkB kinase (IKK) , thus results in
antiapoptotic effects. AKT pathway also involves in chemoresistance.
AKT2 inhibition abrogates gemcitabine-induced activation of
AKT2 and NF-kB, and enhances gemcitabine-induced PUMA
(p53-upregulated modulator of apoptosis) upregulation, resulting
in chemosensitization of pancreatic cancers to gemcitabine .
Consistent with these studies, our data indicated that the inhibition
of IGF-1R/AKT pathway might partly account for the effects of
miR-497 on pancreatic cancer cells. However, miR-497 can also
attenuate the malignancy of cancers by regulating other targets,
such as TARBP2, DICER, BCL2, CCND1, CCNE1, CDC25A,
CCND3, CDK4. [23,24,25].
Increased IGF-1R expression has been found in many cancers
 and displays prognostic values . High expression of
IGF1R in human PDAC tissues has also been reported  and
associates with higher tumor grade and poor survival .
However, plasma IGF-1R levels in pancreatic cancer patients have
not been detected. Our study showed that plasma IGF-1R levels in
patients with pancreatic cancer increased significantly, compared
with that in patients with chronic pancreatitis, other pancreatic
tumors and PNET. But there was no significant difference of
plasma IGF-1R levels between pancreatic cancer patients and
healthy volunteers, which might be elucidated partly by that
IGF-1R was generally expressed in normal tissues, such as liver,
endometrium and neural cells . The value of plasma IGF-1R
at diagnosis of pancreatic cancer was evaluated in the current
study. There was a potential value for distinguishing pancreatic
cancer from chronic pancreatitis, PNET and other pancreatic
tumors. But the diagnostic sensitivity or specificity was not perfect,
large sample detections were needed to further identify the
diagnostic value of plasma IGF-1R.
TNM stage was associated with plasma IGF-1R levels in our
study. Patients with advanced stage tumors had high levels of
plasma IGF-1R. Plasma IGF-1R could be a new biomarker for
guiding TNM stage of pancreatic cancer. Surprisingly, high
expression of plasma IGF-1R was not an adverse prognostic factor
in the present study.
MiR-497 was significantly downregulated in pancreatic cancer
tissues. Upregulation of miR-497 suppressed the malignancy of
pancreatic cancer and re-sensitized PDAC cells to gemcitabine by
directly downregulating IGF-1R protein expression. Plasma
IGF1R levels in pancreatic cancer patients increased, and displayed
potential values for distinguishing pancreatic lesions. IGF-1R
could be also a new plasma biomarker for guiding TNM stage of
Figure S1 The expression level of miR-497 after
transfection of mimics or inhibitor. MiR-497 expression was
detected by qRT-PCR. U6 was served as an internal control. (A)
BxPC-3 cells transfected with miR-497 mimics showed an increase
in miR-497 expression. (B) Cells transfected with miR-497
inhibitor showed a decrease in miR-497 expression. Data were
shown as mean6SD. (* P,0.05).
Figure S2 Relative expression levels of cleaved
caspase3 and PARP. Data were shown as mean6SD. (A) The relative
expression levels of proteins in AsPC-1 cells. (B) The relative
expression levels of proteins in BxPC-3 cells. (* P,0.05).
Figure S3 Relative expression levels of IGF-1R and
pAKT. Relative expression levels were shown as mean6SD. (A)
The relative levels of IGF-1R and p-AKT in AsPC-1 cells. (B) The
relative levels of proteins in BxPC-3 cells. (* P,0.05).
Conceived and designed the experiments: JWX TPZ YPZ. Performed the
experiments: JWX TXW LY. Analyzed the data: JWX LFZ HS.
Contributed reagents/materials/analysis tools: TXW LY HS. Wrote the
paper: JWX TPZ. All authors read and approved the final manuscript:
JWX TXW LY LFZ HS TPZ YPZ.
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