HNRNPA2B1 regulates the epithelial–mesenchymal transition in pancreatic cancer cells through the ERK/snail signalling pathway
Dai et al. Cancer Cell Int
HNRNPA2B1 regulates the epithelial- mesenchymal transition in pancreatic cancer cells through the ERK/snail signalling pathway
Shengjie Dai 0 1 3
Jie Zhang 1 3
Shihao Huang 1 3
Bin Lou 1 3
Binbo Fang 1 3
Tingting Ye 1 3
Xince Huang 1 3
Bicheng Chen 0 1 2 3
Mengtao Zhou 0 1 3
0 Mengtao Zhou, Bicheng Chen and Shengjie Dai contributed equally to this work
1 Department of Surgery, The First Affiliated Hospital, Wenzhou Medical University , 2 FuXue Lane, Wenzhou 325000, Zhejiang Province , People's Republic of China
2 Zhejiang Provincial Top Key Discipline in Surgery, Wenzhou Key Laboratory of Surgery , Wenzhou, Zhejiang Province , People's Republic of China
3 Department of Surgery, The First Affiliated Hospital, Wenzhou Medical Uni- versity , 2 FuXue Lane, Wenzhou 325000, Zhejiang Province , People's Republic of China
Background: Heterogeneous nuclear ribonucleoprotein A2B1 (HNRNPA2B1) is closely related to tumour occurrence and development, oncogene expression, apoptosis inhibition and invasion and metastasis capacities. However, its function in the epithelial-mesenchymal transition (EMT) of pancreatic cancer is not fully understood. Methods: By comparing various wild-type pancreatic cancer cell lines, we determined which have a higher expression level of HNRNPA2B1 accompanied by the higher expression of N-cadherin and vimentin and lower expression of E-cadherin. Therefore, to elucidate the role of HNRNPA2B1 in EMT, we generated models of HNRNPA2B1 knockdown and overexpression in different types of pancreatic cancer cell lines (MIA Paca-2, PANC-1 and Patu-8988) and examined changes in expression of EMT-related factors, including CDH1, CDH2, vimentin and snail. Results: The results show that HNRNPA2B1 promotes EMT development by down-regulating E-cadherin and upregulating N-cadherin and vimentin, and also stimulates the invasion capacity and inhibits viability in human pancreatic cancer cell lines, the similar results in vivo experiments. Moreover, we found that HNRNPA2B1 likely regulates EMT progression in pancreatic carcinoma via the ERK/snail signalling pathway. Conclusions: The results of this work suggest that HNRNPA2B1 inhibition has potential antitumour effects, which warrants in-depth investigation.
Epithelial-mesenchymal transition; HNRNPA2B1; ERK/snail; Pancreatic cancer
Pancreatic carcinoma, which is nearly almost always
fatal, is the fourth leading cause of cancer-related death
worldwide, with fewer than 7% of patients surviving
more than 5 years . Chemotherapy (mainly based on
gemcitabine and capecitabine) and radiation therapy
remain the mainstay of treatment for pancreatic cancer,
with poor efficacy in terms of extending patient survival
[2, 3]. Additionally, although the curative effect is better,
only 15% of patients who undergo surgery survive more
than 5 years [4, 5]. In view of this dismal outcome, to
enable treatment of this deadly disease with new druggable
targets, there is an urgent need to thoroughly understand
the molecular mechanisms of pancreatic cancer
pathophysiology and development.
Recent studies have shown that heterogeneous nuclear
ribonucleoproteins A2B1 (HNRNPA2B1), two
structurally homologous proteins belonging to the hnRNP family,
play important roles in normal development as well as in
cancer processes in eukaryotic cells . Indeed, elevated
HNRNPA2B1 levels in tumours accelerate pre-mRNA
processing via RNA binding, indicating the critical role of
HNRNPA2B1 in the development of carcinoma. Recent
work shows that the epithelial–mesenchymal transition
(EMT), a process in which epithelial cells transform into
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cells with mesenchymal phenotypes, may be regulated
by HNRNPA2B1. During EMT in tumour cells,
up-regulation of vimentin and N-cadherin and down-regulation
of E-cadherin (cell–cell adhesion molecules) promote
cell invasion and metastasis in various cancers,
including pancreatic cancer [7–10]. Other factors also directly
or indirectly trigger EMT, including claudins, occludin,
fibronectin, twist1, ZEB2 and snail [11–15]. Zhou et al.
 indicated that HNRNPAB induces EMT and
promotes metastasis of hepatocellular carcinoma by
transcriptionally activating snail , and Barcelo et al. 
showed that HNRNPA2B1 plays a key role in
Kras-mutation associated pancreatic cancer . Based on these
findings, HNRNPA2B1 might play an important role in
EMT during tumour development.
To date, however, no study has reported on the role
of HNRNPA2B1 in EMT in pancreatic cancer. Thus, we
aimed to determine the roles and potential application
of HNRNPA2B1 in the EMT of pancreatic cancer by
exploring the impact of HNRNPA2B1 knockdown and
overexpression on EMT and the subsequent invasion
and metastasis of pancreatic cancer cells. Furthermore,
we here demonstrate the possibility that HNRNPA2B1
regulates and controls EMT in pancreatic cancer cells
through the ERK/snail pathway. In conclusion, we
conclude that HNRNPA2B1 plays a critical role in pancreatic
The human pancreatic cancer cell lines used in this study
include Patu-8988, MIA Paca-2 and PANC-1. The MIA
Paca-2 and PANC-1 lines were obtained from the
Institute of Biochemistry and Cell Biology, the Chinese
Academy of Science (Shanghai, People’s Republic of China),
and Patu-8988 cells were provided by Genechem
(Shanghai, China). Patu-8988 cells were cultured in RPMI-1640
medium, and MIA Paca-2 and PANC-1 cells were
cultured in DMEM. All media contained 10% foetal bovine
serum, penicillin (100 U/mL), and streptomycin (100 μg/
mL), and the cells were incubated at 37 °C with 5% CO2.
Cell were harvested and passaged at approximately
80–100% confluence using phosphate-buffered saline
(PBS) with 0.25% trypsin and 0.01% EDTA.
Reagents and antibodies
Antibodies against HNRNPA2B1, E-cadherin, vimentin,
N-cadherin, MMP7, MMP9, ERK1/2 and
phosphorylated proteins were purchased from Abcam (Cambridge,
USA). An antibody against β-actin was purchased from
Bio-world Technology (St Louis Park, MN, USA). An
antibody against snail was purchased from Cell
Signaling Technology (Danvers, USA). Foetal bovine serum
(FBS) was purchased from Sigma Chemical (St Louis,
MO, USA). Roswell Park Memorial Institute
(RPMI)1640 medium, Dulbecco’s Modified Eagle’s Medium
(DMEM), and trypsin were purchased from GIBCO
(Grand Island, NY, USA). ERK-inhibitor (GSK2656157)
was purchased from MedChem Express (New Jersey,
USA) and gemcitabine was purchased from Jkchem
Knockdown and overexpression of HNRNPA2B1
A lentivirus plasmid containing a short hairpin RNA
(shRNA) for HNRNPA2B1(KH)and its negative control
(NKH) and a lentivirus plasmid containing HNRNPA2B1
driven by the CMV promoter (OH) and its negative
control (NOH) were designed and produced by
Genechem (Shanghai, China). For transfection, MIA
Paca2, PANC-1 and Patu-8988 cells were seeded in 6-well
plates at 5 × 104 cells and allowed to attach until nearly
30–40% confluent. After removal of the culture medium,
the plates were washed three times with PBS, and
lentivirus and 50 μg/mL polybrene was added to each plat.
After 12 h of transfection, the transfection medium was
replaced with standard medium. Cells were harvested for
passaging or testing when they occupied 80% of the plate.
All procedures were performed using biohazard safety
Western blot analysis
Protein was extracted by adding RIPA buffer (Beyotime,
Shanghai, China) with protease inhibitor (Beyotime,
Shanghai, China) and phosphatase inhibitor (Roche
Diagnostics GmbH, Mannheim, Germany). The final
protein lysate was centrifuged, and the supernatant was
collected. BCA (Beyotime, Shanghai, China) was used to
measure the protein concentration. After denaturing, the
protein mixture was separated using a 10%
polyacrylamide gel; a prestained protein marker was included. The
separated proteins were transferred to polyvinylidene
difluoride (PVDF) membranes, which were blocked with 5%
skim milk in Tris-buffered saline (10 mmol/L Tris–HCl,
pH 8.0, containing 150 mmol/L NaCl and 0.1%
Tween20) for 2 h at room temperature before being incubated
at 4 °C overnight with primary antibodies diluted in First
Ab Dilute. The membranes were then incubated with
horseradish peroxidase (HRP)-conjugated secondary
antibodies (Bio-world Technology, MN, USA) for 2 h at
room temperature after washing with TBST. The
densities of the protein bands were recorded and measured by
RNA extraction and quantitative real‑time PCR analysis
When the cells were approximately 90% confluent, total
RNA was extracted using TRzol Reagent (Ambion,
Carlsbad, California, USA) according to the
manufacturer’s instructions. To obtain cDNA, reverse
transcription was performed with RevertAid First Strand cDNA
Synthesis Kit(Thermo, Manassas, USA). qRT-PCR was
performed with SYBR Green Master (Biosystems,
Foster City, CA, USA) using a 7500 Real-Time PCR System
(Applied Biosystems, Foster City, USA). β-Actin was
amplified as an internal standard, and ΔCt values were
used for analysis of the quantitative real-time PCR data.
Polymerase chain reaction (PCR) primers were
purchased from Synbio Tech (Jiangsu, People’s Republic of
China); the sequences are listed in Table 1.
Cell viability assay
The different cell models were plated into 96-well plates
containing 1 mL 10% FBS medium at a density of 10,000
cells per well and incubated for 24 h. The cells were
starved in FBS-free medium for 12 h when the cells in
each well reached 70% confluence. The medium was
discarded, and the cells were washed once with PBS before;
100 μL FBS-free medium containing 10 μL Cell Counting
Kit 8 (CCK8; Dojindo, Kuma-moto, Japan) solution was
added to each well and incubated for 1–2 h. Cell viability
is expressed as the fold change in absorbance at 490 nm
for each well, as measured using a microplate reader
(BioTek, Winooski, VT, USA).
Cell invasion assay
Transwell Permeable Supports (Costar, Kennebunk,
USA) with matrigel Basement Membrane Matrix
(Corning, Franklin Lake, NJ, USA) bedding were used with an
8 μm porosity polyethylene terephthalate membrane.
Cells (5 × 104) were starved overnight and added to
the upper chamber in 200 μL FBS-free medium; 500 μL
β-actin GACATCCGCAAAGACCTG GGAAGGTGGACAGCGAG
HNRNPA2B1 ATGGCTGCAAGACCTCATTC TAATTCCGCCAACAAACAGC
E-cadherin GACCGAGAGAGTTTCCCT GGTGGGATTGAAGATCGGAG
N-cadherin GTGACCGATAAGGATCA TTGACCACGGTGACTAACCC
Vimentin GGATGTTGACAATGCGTCTC CTCCTGGATTTCCTCTTCGT
Mmp7 TGAGGATGAACGCTGGACG CACTGCATTAGGATCAGAGG
Mmp9 AGTCCACCCTTGTGCTCTTC GCCACCCGAGTGTAACCAT
ERK ATCCCCATCACAAGAAGA GCTTTGGAGTCAGCATTT
Snail CTTCTCCTCTACTTCAGT TGAGGTATTCCTTGTTGCAGT
RPMI-1640 (for Patu-8988 cells) or DMEM (for PANC-1
and MIA Paca-2 cells) with 10% FBS was added to the
lower compartment as a chemoattractant. The cells were
incubated for 24 h, and the filters were washed three
times with PBS, fixed with 4% paraformaldehyde for
30 min, stained with 4 g/L crystal violet for 15 min and
washed twice with PBS. The cells on the upper surface of
the filter were gently scraped off. The stained cells in
randomly selected fields were observed and counted using a
200× inverted microscope.
Cell cycle analysis
The cell cycle analysis was measured using the
Propidium iodide Kit for Flow Cytometry (Multiscience,
Hangzhou, Zhejiang, China), according to the manufacturer’s
instructions. After the treatments, cell cycle analysis was
measured using a Thermo Attune and analyzed using the
ModFit LT2.0 software (Coulter, Miami, FL).
Establishment of the xenografted tumor model in nude
Athymic nude mice (BALB/C-nu/nu, 6–8 weeks old,
female) were gained from the Animal Center of Chinese
Academy of Science (Shanghai, China) and fed under
specific pathogen-free conditions in the laboratory
animal center of Wenzhou Medical University (Wenzhou,
Zhejiang, China). We established a pancreatic
xenografted tumor model through subcutaneous injection
of 1 × 107 pancreatic cancer cells per mouse. After the
treatments, we observe the growth rate of tumor volume
and the change of weight on each mice. All researches
involving animals were approved by the Animal Ethics
Committee of Wenzhou Medical University.
Data are expressed as the mean ± standard error of the
mean (SEM) for at least three independent experiments.
Analysis of variance was utilised to distinguish
differences in each group. A value of P < 0.05 was considered
to be statistically significant. All analyses were performed
using SPSS 16.0 software (IBM, USA).
HNRNPA2B1 expression was associated with the
mesenchymal phenotype in pancreatic cancer cell lines
We first explored the relationship between HNRNPA2B1
and EMT in wild-type human pancreatic cancer cell lines
(Patu-8988, MIA Paca-2 and PANC-1) by qRT-PCR and
Western blotting and we found highest N-cadherin and
HNRNPA2B1 expression and lowest E-cadherin in the
Patu-8988 cell line compared to the Panc-1 and MIA
Paca-2 cell line. The expression levels of HNRNPA2B1 in
the different cells was directly correlated to the levels of
N-cadherin and vimentin and inversely correlated to the
levels of E-cadherin (P < 0.05; Fig. 1a, b).
HNRNPA2B1 regulated cell viability and EMT in pancreatic
cancer cell lines
We then generated cell models of HNRNPA2B1
knockdown and overexpression and examined accompanying
changes in cell viability. All cell lines were treated with
trypsinisation, and the cells in suspension were assayed
using CCK8 after 24 h of incubation. Non-transfected
pancreatic carcinoma cell lines were used as controls.
The notable linearity of the graph revealed the lowest
viability for the HNRNPA2B1 knockdown cells.
Moreover, the non-transfected cells and the HNRNPA2B1
lentivirus negative control cells showed viability between the
knockdown and overexpressing cells, with no statistical
significance between them (P > 0.05; Fig. 1c). The level of
HNRNPA2B1 expression was correlated with cell
viability. To investigate if the cell cycle analysis is conforming
to the consequence of CCK8 in the pancreatic cancer
cell lines with HNRNPA2B1 knockdown or
overexpression were generated by lentiviral gene delivery system.
We were able to detect the G0G1 cell cycle arrest of
Patu8988-KH, Panc-1-KH and MIA Paca-2-KH cells and the
S-Phase cell cycle increased of Patu-8988-OH,
Panc1-OH and MIA Paca-2-OH cells, but no significance
difference was found in the G2M cell cycle of those cells
(Fig. 2). So we concluded that HNRNPA2B1 could
promote the proliferation of pancreatic cancer cells.
We then sought insight into the relationship between
HNRNPA2B1 and EMT. In addition to HNRNPA2B1,
we also analysed EMT markers (E-cadherin, N-cadherin
and vimentin). Western blotting assays showed that the
protein levels of HNRNPA2B1 were decreased to 66%
in Patu-8988, 52% in MIA Paca-2 and 50% in PANC-1
cells with knockdown but were increased to 1000% in
Patu-8988, 246% in MIA Paca-2 and 160% in PANC-1
with overexpression. A similar pattern was observed by
qRT-PCR (Fig. 3a). When HNRNPA2B1 was knocked
down, the messenger RNA (mRNA) and protein levels
of CDH1 (E-cadherin) increased, whereas these levels
were decreased when HNRNPA2B1 was overexpressed
(Fig. 3b). As expected, N-cadherin (Fig. 3c) and vimentin
(Fig. 3d) showed trends opposite to that of E-cadherin.
Furthermore, we confirmed that lower HNRNPA2B1
expression corresponded with higher E-cadherin
expression and lower N-cadherin and vimentin expression in
pancreatic cancer cells.
What’s more, we treated Patu-8988 cells with
gemcitabine in 40, 80 and 160 µmol/L tentatively, and found
the inhibiting effect of 160 μmol/L group is the strongest
(Fig. 7a). Then, we respectively treated Patu-8988-OH,
Panc-1-OH and MIA Paca-2-OH cells with gemcitabine
in 160 μmol/L and discovered gemcitabine could inhibite
EMT similar to HNRNPA2B1 (Fig. 7b, e). In conclusion,
HNRNPA2B1 could regulated cell viability, EMT and the
HNRNPA2B1 promoted invasion in pancreatic carcinoma
Because of the biological phenotype resulting from EMT,
we evaluated the capacity of cells to invade to confirm
whether HNRNPA2B1 is directly or indirectly associated
with EMT. As shown by Fig. 4c and d, transwell
invasion assays indicated a decrease in the invasion rate of
KH cells but an increase in the invasion rate of OH cell.
According to the results, HNRNPA2B1 could stimulate
cell invasion after accelerating the development of EMT.
We also tested expression of MMPs under
different conditions because MMPs enhance cell invasion by
degrading the extracellular matrix. qRT-PCR and
Western blotting showed significant decreases in the
cellular levels of Mmp7 (Fig. 4a) and Mmp9 (Fig. 4b) after
HNRNPA2B1 knockdown and increases after
HNRNPA2B1 overexpression. These results are consistent with
the invasion results.
Furthermore, we established xenografted tumor in
nude mouse via subcutaneous injection of 1 × 107
PANC-1, PANC-1-KH and PANC-1-OH cells per mouse
severally. As shown by Fig. 5a–c, we observed that the
largest tumor volume and the thinnest in the
PANC-1OH-mice but an antipodal phenomenon in the
PANC1-KH-mice. Also, we found the tumor inhibitory rate of
the PANC-1-KH group is the highest by using the
PANC1-OH group as a reference substance due to its
highest expression of HNRNPA2B1 (Fig. 5d). These data are
consistent with that HNRNPA2B1 could stimulate the
growth and invasion in pancreatic cancer.
HNRNPA2B1 regulated EMT progression via the ERK/snail
pathway in pancreatic cancer cell lines
Splicing factor hnRNP A2 has been shown to activate the
RAS-MAPK-ERK pathway by integrating A-RAF
splicing signalling . In addition, ERK activation
markedly up-regulates expression of snail, one of the main
transcription factors influencing EMT [19–25]. When
HNRNPA2B1 was knocked down, snail expression was
decreased according to both qRT-PCR and Western
blotting (Fig. 6c), and that p-ERK1/2 expression was
decreased according to Western blotting (Fig. 6b). In
contrast, nonphosphorylated ERK1/2 remained at a higher
level (Fig. 6a). The results obtained when HNRNPA2B1
was overexpressed were opposite to the results obtained
Fig. 1 HNRNPA2B1 improves cell viability, and its expression was associated with EMT markers in pancreatic cancer cell lines. The mRNA expression
levels of HNRNPA2B1 and EMT representative markers (E-cadherin and N-cadherin) in Panc-1 and Patu-8988 pancreatic cancer cells are presented as
a histogram (a); HNRNPA2B1, E-cadherin, N-cadherin and vimentin were visualised by Western blotting (b); data represent the mean ± SEM, n = 3,
*denotes P < 0.05 vs Patu-8988 groups. Fold change in cell viability of Patu-8988, PANC-1 and MIA Paca-2 cells (c); data represent the mean ± SEM,
n = 3, *denotes P < 0.05 vs NC groups, **denotes P < 0.05 vs Patu-8988 cells, ***denotes P < 0.05 vs Panc-1 cells, #means P > 0.05 vs NC groups
when HNRNPA2B1 was knocked down. And then we
respectively treated Patu-8988-OH, Panc-1-OH and MIA
Paca-2-OH cells with ERK inhibitor, we observed that
EMT was inhibited in the treated groups comparing with
control groups (Fig. 7c, d, f ). Therefore, we tentatively
conclude that HNRNPA2B1 regulates EMT progression
via the ERK/snail pathway in pancreatic cancer cell lines.
Pancreatic cancer, which currently has the worst
prognosis, is the most common malignant tumour of the
digestive system and among the top ten malignant tumours
in our country . Although surgery is the only radical
treatment presently available, the curative rate of
pancreatic cancer remains very low because surgery is often no
longer an option at the time of diagnosis .
Accordingly, biotherapy treatments have become more popular
over the past few years. Encouraging results have been
reported by recent studies showing that pancreatic
carcinoma could be inhibited by controlling several
biomarkers such as SMAD4, CXCR2, ABCG2 and Kras [28–34].
In addition, Barcelo et al. showed that HNRNPA2B1
interacts with and regulates oncogenic Kras in pancreatic
ductal adenocarcinoma cells, and Tauler et al. indicated
Fig. 2 HNRNPA2B1 changes cell cycle in pancreatic cancer cell lines. The cell cycle of normal pancreatic cancer cell lines (Patu-8988, MIA Paca-2 and
PANC-1) and its treated cells were visualised by flow cytometry (a). And a histogram of the proportion for cell cycle were drew in b. Data represent
the mean ± SEM, n = 3, *denotes P < 0.05 vs NC groups, #denotes P > 0.05 vs NC groups
Fig. 3 HNRNPA2B1 increases EMT in pancreatic cancer cell lines. HNRNPA2B1 (a), E-cadherin (b), N-cadherin (c) and vimentin (d) were visualised by
qRT-PCR and Western blotting (histograms of mRNA expression on pictures of protein levels). β-Actin was used as an internal control. Data
represent the mean ± SEM, n = 3, *denotes P < 0.05 vs NC groups, #denotes P > 0.05 vs NC groups
that HNRNPA2B1 modulates EMT in lung cancer cell
lines [17, 35]. However, a clear relationship between
HNRNPA2B1 and EMT in pancreatic cancer and the
signalling pathway involved remain elusive.
We previously discovered that the level of
HNRNPA2B1 expression is higher in pancreatic cancer than in
non-lesion tissue, as are other cancers, such as lung
cancer , hepatocellular carcinoma  and glioblastoma
. Based on our study, we chose several pancreatic
carcinoma cell lines to simulate different types of pancreatic
cancers and generated models of HNRNPA2B1 depletion
and overexpression using Crispr/Cas9 genetic
technology. Unsurprisingly, conforming to the manifestation
of an oncogene, both the mRNA and protein levels of
HNRNPA2B1 were closely related with EMT, which plays
an important role in the invasion and metastasis of
cancer cells [38, 39]. Moreover, we found that the ERK/snail
signalling pathway, regulated by HNRNPA2B1, plays a
crucial role in pancreatic carcinoma.
EMT has a vital function in the process of phenotypic
conversion [15, 40–42]. Accumulating experimental
evidence suggests that many pancreatic cancer cell lines
express higher levels of mesenchymal biomarkers, such
as vimentin and N-cadherin, and lower levels of
epithelial biomarkers, such as E-cadherin, than normal
pancreatic cell lines. In our study, we found that the cell lines
expressing higher levels of HNRNPA2B1 had enhanced
invasive and migration capacities as well as lower
expression of E-cadherin and higher expression of vimentin
and N-cadherin than the others cells. Moreover, we
detected levels of MMP7 and MMP9 expression and
found that HNRNPA2B1 promoted expression of these
MMPs, with a correlated change in of MMP expressions
and EMT development. In addition, transwell
invasiveness assays showed a greater number of cells passing
through the membrane when MMP levels were higher
due to extracellular matrix degradation. What’s more,
vivo study showed that the cells with higher expression
of HNRNPA2B1 could induce bigger xenografted tumor
in mice. Therefore, we speculate that HNRNPA2B1 may
be a potent inducer of EMT in pancreatic carcinoma.
Meanwhile, snail, an important transcription factor
Fig. 4 HNRNPA2B1 enhances cell invasion and expression of MMPs in pancreatic cancer cell lines. Mmp7 (a) and Mmp9 (b) were visualised by
qRTPCR and Western blotting (histograms of mRNA expression on pictures of protein levels). β-Actin was used as an internal control. The cell invasion
ability c was evaluated in normal cells, cells transfected with a negative lentivirus control and cells transfected with a lentivirus. Cells were counted
in 3 randomised 200× fields for invasion (d). Data represent the mean ± SEM, n = 3, *denotes P < 0.05 vs NC groups, #denotes P > 0.05 vs NC
up-regulation in EMT, is noteworthy because snail
triggers EMT by coordinating the induction of mesenchymal
biomarkers and the repression of epithelial biomarkers,
which could induce EMT and promote metastasis of
hepatocellular carcinoma. We found that the expression
level of snail mRNA and protein consistently increased
when HNRNPA2B1 was overexpressed. However, the
concrete mechanism by which this occurs in pancreatic
cancer requires further investigation.
As one of the most important intracellular pathways,
RAS/RAF/MEK/ERK signalling causes multiple changes
in the expression of various genes, transmitting signals
from receptors to regulate gene expression and prevent
apoptosis [43–46]. Recent studies have shown that
activation of the ERK pathway, not only by growth factors but
also by mutations occurring in cancer cells in the genes
encoding RAS or RAF, contributes to EMT. RAS and
RAF signalling also activate expression of snail1 and/
or snail2, therefore promoting cell motility and invasive
behaviour in cancer-associated EMT . Moreover,
Hsu et al. showed that snail promotes cell motility and
invasive behaviour in cancer-associated EMT by
activating the ERK signalling , HNRNPA2B1, a member of
a heterogeneous group of nuclear ribonucleoproteins,
has been shown to contribute to ERK activation by
controlling A-RAF splicing . We explored the
relationship between snail and the ERK pathway and found that
the expression level of snail was notably decreased with
attenuation of ERK phosphorylation caused by
HNRNPA2B1 knockdown. Futher more, when we inhibited the
activation of ERK1/2, both of HNRNPA2B1 and EMT
were decreased. Therefore, we tentatively conclude that
HNRNPA2B1 promotes EMT by activating the ERK/
snail pathway in pancreatic cancer, revealing a potential
Fig. 5 HNRNPA2B1 promotes the growth rate of xenografted tumor and decreases the weight in vivo. 1 × 107 Panc-1, Panc-1-OH and Panc-1-KH
cells were injected in subcutaneous tissues per athymic nude mouse. Mouse were put in the blue drape for photograph on day 0, day 10 and day
25 severally (a). The body weight (b) and tumor volume (c) of each mouse was measured and visualized as line graphs. Tumor inhibitory rate was
calculated by using the Panc-1-OH group as a reference substance (d). Data represent the mean ± SEM, n = 3, *denotes P < 0.05 vs NC groups,
**denotes P < 0.05 vs Panc-1-OH group
Fig. 6 HNRNPA2B1promotes EMT via ERK/snail signalling in pancreatic cancer cell lines. Nonphosphorylated ERK1/2 (a), phospho-ERK1/2 (b) and
snail (c) were visualised by qRT-PCR and Western blotting (histograms of mRNA expression on pictures of protein levels). β-Actin was used as an
internal control. Data represent the mean ± SEM, n = 3, *denotes P < 0.05 vs NC groups, #denotes P > 0.05 vs NC groups
connection between HNRNPA2B1 and EMT.
Nonetheless, the mechanism of specific subunit by which
HNRNPA2B1 affects the intracellular RAS/RAF/MEK/ERK
cascade is still unknown, and the mechanism by which
HNRNPA2B1 transfers a signal to various pathways and
which subunits are involved requires further investigation.
Fig. 7 ERK inhibitor and gemcitabine inhibite HNRNPA2B1 and EMT in pancreatic cancer cell lines. The expression of HNRNPA2B1 in Patu-8988 cells
treated with gemcitabine in 40, 80 and 160 μmol/L were visualised by Western blotting (a). The expression of HNRNPA2B1, E-cadherin, N-cadherin,
Vimentin, ERK1/2, p-ERK1/2 and snail in Patu-8988-OH, Panc-1-OH and MIA Paca-2-OH cells treated with gemcitabine in 160 μmol/L were visualised
by qRT-PCR (e) and Western blotting (b). The expression of HNRNPA2B1, E-cadherin, N-cadherin, Vimentin, ERK1/2, p-ERK1/2 and snail in
Patu8988-OH, Panc-1-OH and MIA Paca-2-OH cells treated with ERK inhibitor were visualised by qRT-PCR (c, d) and Western blotting (f). β-Actin was used
as an internal control. Data represent the mean ± SEM, n = 3, *denotes P < 0.05 vs control groups
In summary, our study shows that via ERK/snail
pathway, HNRNPA2B1 might increase invasion ability by
activating EMT phenotypes in pancreatic cancer. Our
results not only provide a basis for establishing
HNRNPA2B1-targeted molecular therapy in pancreatic cancer
but also enrich the current understanding of
HNRNPA2B1’s regulation of EMT and its potential signalling
EMT: epithelial–mesenchymal transition; NC: normal control; KH: cells
transfected with HNRNPA2B1 shRNA lentivirus; NKH: cells transfected with
HNRNPA2B1 knockdown negative control lentivirus; OH: cells transfected with
HNRNPA2B1 overexpression lentivirus; NOH: cells transfected with HNRNPA2B1
overexpression negative control lentivirus; SEM: standard error of the mean;
MMPs: matrix metalloproteinases.
DSJ carried out the molecular genetic studies, participated in the Western
blot analysis and drafted the manuscript. ZJ, LB and HXC carried out the cell
viability assay and cell invasion assay. HSH and YTT participated in the design
of the study and performed the statistical analysis. FBB performed the study
in vivo experiment. ZMT and CBC conceived of the study, and participated in
its design and coordination and helped to draft the manuscript. All authors
read and approved the final manuscript.
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