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Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling pathway regulates ABC transporters
to reverse gemcitabine resistance in pancreatic cancer
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* Published: 10 May 2022

HSA-MIR-3178/RHOB/PI3K/AKT, A NOVEL SIGNALING PATHWAY REGULATES ABC TRANSPORTERS
TO REVERSE GEMCITABINE RESISTANCE IN PANCREATIC CANCER

* Jianyou Gu1,2 na1,
* Wenjie Huang1,2 na1,
* Xianxing Wang2,
* Junfeng Zhang2,
* Tian Tao1,
* Yao Zheng2,
* Songsong Liu2,
* Jiali Yang2,
* Zhe-Sheng Chen3,
* Chao-Yun Cai3,
* Jinsui Li1,
* Huaizhi Wang2 &
* …
* Yingfang Fan1

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Molecular Cancer volume 21, Article number: 112 (2022) Cite this article

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ABSTRACT

BACKGROUND

Although gemcitabine has been considered as the first-line drug for advanced
pancreatic cancer (PC), development of resistance to gemcitabine severely limits
the effectiveness of this chemotherapy, and the underlying mechanism of
gemcitabine resistance remains unclear. Various factors, such as ATP binding
cassette (ABC) transporters, microRNAs and their downstream signaling pathways
are included in chemoresistance to gemcitabine. This study investigated the
potential mechanisms of microRNAs and ABC transporters related signaling
pathways for PC resistance to gemcitabine both in vivo and in vitro.

METHODS

Immunohistochemistry and Western blotting were applied to detect the expression
of ABC transporters. Molecular docking analysis was performed to explore whether
gemcitabine interacted with ABC transporters. Gain-of-function and
loss-of-function analyses were performed to investigate the functions of
hsa-miR-3178 in vitro and in vivo. Bioinformatics analysis, Western blotting and
dual-luciferase reporter assay were used to confirm the downstream regulatory
mechanisms of hsa-miR-3178.

RESULTS

We found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant
PC tissues and cells. Molecular docking analysis revealed that gemcitabine can
bind to the ABC transporters. Hsa-miR-3178 was upregulated in gemcitabine
resistance PANC-1 cells as compared to its parental PANC-1 cells. Moreover, we
found that hsa-miR-3178 promoted gemcitabine resistance in PC cells. These
results were also verified by animal experiments. RhoB was down-regulated in
gemcitabine-resistant PC cells and it was a downstream target of hsa-miR-3178.
Kaplan–Meier survival curve showed that lower RhoB expression was significantly
associated with poor overall survival in PC patients. Rescue assays demonstrated
that RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance.
Interestingly, hsa-miR-3178 promoted gemcitabine resistance in PC by activating
the PI3K/Akt pathway-mediated upregulation of ABC transporters.

CONCLUSIONS

Our results indicate that hsa-miR-3178 promotes gemcitabine resistance via
RhoB/PI3K/Akt signaling pathway-mediated upregulation of ABC transporters. These
findings suggest that hsa-miR-3178 could be a novel therapeutic target for
overcoming gemcitabine resistance in PC.

INTRODUCTION

Pancreatic cancer (PC) is a malignant tumor with a very poor prognosis. Its
five-year survival rate is only around 10% [1]. Gemcitabine is the cornerstone
drug of PC treatment in all stages of this disease [2]. However, therapeutic
effect of gemcitabine is hampered by drug resistance [3]. The mechanisms of
gemcitabine resistance in pancreatic cancer are complex. Whether gemcitabine
resistance was associated with the ATP-binding cassette (ABC) transporters is
not fully studied [4]. ABC transporters are a superfamily consisting of seven (A
to G) subfamilies with a total of 48 members, and the overexpression of these
transporters is the main mechanism leading to multidrug resistance (MDR) of
tumor chemotherapy [5]. However, little is known about their role in gemcitabine
resistance.

Recent studies on the involvement of microRNAs (miRNAs) in chemoresistance may
offer new opportunity to overcome the chemotherapeutic resistance of gemcitabine
in PC [6,7,8]. MiRNAs are a category of small endogenous single-stranded RNAs
usually play a negative regulatory role by targeting specific mRNAs for
degradation or translation suppression [9, 10]. Aberrant expression of miRNAs is
linked to pathological processes in various cancers, such as cell proliferation,
apoptosis, invasion, metabolism, differentiation and drug resistance
[11,12,13,14,15]. In our previous study, four differentially expressed miRNAs,
including hsa-miR-3178, hsa-miR-485-3p, hsa-miR-574-5p, and hsa-miR584-5p, were
identified in gemcitabine-resistant PC cells versus parental cells. Among them,
hsa-miR-3178 significantly affected the survival of pancreatic cancer patients
(HR = 2.27, Logrank P = 0.0018) [16], but its downstream signaling pathway and
the underlying mechanism remain unclear.

Ras Homolog Family Member B (RhoB) belongs to the small GTPases family. The Rho
subfamily of RhoGTPases plays critical roles in physiological and pathological
processes of cancers, and consists of three proteins, RhoA, RhoB, and RhoC [17].
RhoB is localized at endosomes as well as at the plasma membrane, endosomes,
Golgiassociated vesicles and the nucleus[18, 19]. RhoB is an immediate early
response gene that is induced by a variety of stimuli [18]. RhoB has been
reported to be targeted by various miRNAs to regulate apoptosis, proliferation
and migration of cancer cells [20, 21]. Moreover, as a tumor suppressor,
downregulation of RhoB is associated with drug resistance in cancers [22, 23].
In pancreatic cancer, the relationship between RhoB and gemcitabine resistance
remain elusive.

In this study, we found that RhoB is one of the downstream target molecules of
hsa-miR-3178. Hsa-miR-3178/RhoB axis upregulates the expression of ABC
transporters via activating PI3K/Akt signaling pathway to induce gemcitabine
resistance in PC.

MATERIALS AND METHODS

CELL CULTURES AND REAGENTS

Pancreatic cancer cell lines, AsPC-1, BxPC-3, CFPAC-1, MIA PaCa2, SW1990
(Shanghai Institute of Biochemistry and Cell Biology, China), and Hs766t (ATCC,
Manassas, VA, USA), and an immortalized human pancreatic ductal epithelial cell
line: HPDE6-C7 (BeNa Culture Collection, China), were used in this study. The
acquisition and culture of PC gemcitabine resistant cells (PANC-1-GEM) and their
parental sensitive cells (PANC-1) was the same as described in our previous
article [16]. The PI3K inhibitor LY294002 (10 μM, MCE, USA) and PI3K agonist
740Y‐P (30 μM, MCE, USA) were used to treat cells for 24 h. Gemcitabine were
purchased from Selleck Chemicals Co. Ltd (Houston, TX, USA).

TISSUE SPECIMENS AND IMMUNOHISTOCHEMISTRY (IHC)

The expression of ABC transporter proteins was analyzed by immunohistochemical
staining (IHC). A tissue microarray containing 87 cases of pathologically
diagnosed PC and 3 gemcitabine-resistant PC tissues and 3 gemcitabine-sensitive
tissues were used. Another tissue microarray containing 70 PC and 58 adjacent
nontumor tissues were used. Additionally, 15 pairs of primary fresh-frozen PC
tissue samples and matched adjacent non-tumor tissues, 13 fresh PC tissues and
non-tumor tissues were taken for qRT-PCR analysis. The tissues were obtained
from the Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Army
Medical University in Chongqing, China. The ethics committee of Southwest
Hospital approved the use of the clinical specimens.

IHC was performed according to the manufacturer’s instructions (Maixin, Fuzhou,
China). The IHC score was calculated on the basis of staining intensity and
percentage of positively stained cells. The staining intensity was scored as
follows [24]: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3
(strong staining). The percentage of positively stained was scored as follows: 0
(no staining), 1 (1–10%), 2 (10–50%), 3 (more than 50%). The staining index (SI)
was calculated by multiplying the values of staining intensity and percentage of
positively stained cells. Using this method of assessment, tumor samples were
divided into groups with low (⩽ 4) or high expression (⩾ 6) of the target
proteins.

WESTERN BLOTTING

Western blotting was performed as previously described [25] using antibodies
against RhoB (1:500; Proteintech, USA), phospho-Akt 308, phospho-Akt 473 and Akt
(1:1,000; Cell Signaling Technology, USA), phospho-PI3K (1:1,000; Abcam, USA),
PI3K (1:1,000; Cell Signaling Technology, USA), P-glycoprotein (P-gp, also known
as ABCB1) (1:500; Proteintech, USA), breast cancer resistance protein (BCRP,
also known as ABCG2) (1:500; Proteintech, USA), multidrug resistance-associated
protein 1 (MRP1, also known as ABCC1) (1:500; Abcam, USA) and β-actin (1:5,000;
Cell Signaling Technology, USA). The secondary antibody used is horseradish
peroxidase-conjugated antibody (anti-rabbit; 1:5,000; Cell Signaling
Technology). Protein levels were normalized to the endogenous control β-actin.

IN SILICO MOLECULAR DOCKING ANALYSIS

The structure of gemcitabine was optimized for docking analysis using the
Maestro V11.1 (Schrodinger, LLC, New York, 2020). The hydrogenation and force
field structure were optimized by removing water molecules from the P-gp (PDB
ID: 6FN1) [26], BCRP (PDB ID: 6FFC) [27] and MRP1 (PDB ID: 5UJA) [28] receptor
model structures. The Glide module of Maestro software was used to dock the
protein receptor and ligand in an induced-fit method. The cavity of 20 Å was
selected as the docking active region, and the docking calculation was carried
out with standard parameters. The method of induced-fit can make the gemcitabine
adopt an optimal conformation to bind to the protein receptor, and the protein
receptor will also change the original conformation to better bind to the
gemcitabine [29]. The docking models with the highest score were analyzed and
visualized.

RNA EXTRACTION AND REAL-TIME PCR

Methods and reagents for RNA extraction and qRT-PCR experiments were the same as
described in our previous article [16]. The bulge-loop miRNA qRT-PCR primer sets
specific for hsa-miR-3178 were designed and synthesized by RiboBio Inc
(Guangzhou, China). The RhoB primer with sequences 5'-GCGGTAGGCGTGTACGGT-3'
(forward) and 5'-CTGGAATAGCTCAGAGGC-3' (reverse) were synthesized by the
GeneCopoeia Inc (Guangzhou, China).

OVEREXPRESSION AND SILENCING OF GENES

Agomir-3178 (hsa-miR-3178 mimics, 30 nM), mimic negative control (MNC),
antagomir-3178 (hsa-miR-3178 inhibitor, 30 nM), inhibitor negative control
(INC), RhoB siRNA (50 nM), scramble control (Scr) and overexpression Lentiviral
(pReceiver-Lv216-CMV-1Flag-SV40-Puromycin) were obtained from GeneCopoeia Inc
(Guangzhou, China). The transfection reagent used was Lipofectamine 3000
(Invitrogen, USA).

LUCIFERASE REPORTER ASSAY

The wild-type and mutant 3’ UTR sequence of RhoB were inserted into the
SI-Check2 vector at a position downstream of the SV40 promoter. Mutations were
generated in the binding sites of the 3’ UTR. Next, 0.16 µg plasmid containing
the RhoB-3’UTR/RhoB-3’UTR mutant and 5 pmol hsa-miR-3178/Negative Control (NC)
was transfected into 293 T cells, PANC-1 cells and PANC-1-GEM cells. Firefly
luciferase (internal reference) and Renilla luciferase activities at 48 h after
transfection were detected using the Promega Dual-363 Luciferase system
according to the manufacturer’s instructions.

CELL VIABILITY ASSAY

Cell viability was determined by Cell Counting Kit 8 (CCK-8) assay. The specific
steps and reagents used were the same as described in our previous article [16].
Cells were harvested and resuspended, and seeded in a 96-well plate at 5 × 103
cells per well. After incubating for 24 h, gemcitabine was added. After 72 h of
incubation, CCK-8 (10 µl) was added to each well and the cells were further
incubated for 2 h before detection. The half-inhibitory concentration (IC50) of
anticancer drug, at which 50% of cells were inhibited, was calculated as
previously described [30].

CELL PROLIFERATION ANALYSIS

The viability and proliferation of PC cells was determined using the CCK-8 assay
and the 5-Ethynyl-2’-deoxyuridine (EdU) immunofluorescence assay (RiboBio,
Guangzhou, China) after cells were treated with gemcitabine (1 μmol/L, PANC-1
cells; 150 μmol/L, PANC-1 cells) for 72 h. For CCK-8 assay, 5 × 103 cells with
or without transfection were seeded in 96-well plates and 10 µl of Cell Counting
Kit-8 solution were added to each well and incubated for 2 h at 37 °C before
detection. The OD value was detected at 24, 48, 72, and 96 h after the cells
were seeded. For EdU assay, 5 × 103 cells with or without transfection were
seeded in 96-well plates. After incubation for 72 h (gemcitabine, 1 μmol/L,
PANC-1 cells; 150 μmol/L, PANC-1 cells), the cells were incubated with EdU and
immunofluorescence staining was performed.

APOPTOSIS ASSAYS

Cellular apoptosis was quantified by flow cytometry using an Annexin V-FITC/PI
Staining kit according to the manufacturer’s instruction (KeyGEN BioTECH,
Jiangsu, China). Briefly, after treatment with gemcitabine (1 μmol/L, PANC-1
cells; 150 μmol/L, PANC-1 cells) for 72 h, 5 × 105 cells were harvested and
suspended in 500 μl of binding buffer containing 5 μl annexin V-FITC and 5 μl
PI. Then cells were incubated for 15 min at room temperature in the dark. The
apoptotic cells were detected by a FACS Calibur (BD Biosciences).

ANIMAL EXPERIMENTS

Four to six weeks female BALB/c nude mice were purchased from the Peking
University Animal Center (Beijing, China). The mice were randomized into 6
subgroups (n = 4/group). The mice were inoculated subcutaneously in the left
oxter with 5 × 106 PANC-1 cells (transfected with agomir-3178, agomir negative
control or PBS) or PANC-1-GEM cells (transfected with antagomir-3178, antagomir
negative control or PBS). One week after injection, when the mice had a palpable
tumor with a diameter of ~ 2 mm, gemcitabine (5 mg/kg) was administered
intraperitoneally every three days. Tumor length (L) and width (W) were
monitored every week using a digital Vernier caliper and tumor volume was
determined using the equation (L*W2)/2. The mice were sacrificed 4 weeks post
injection and tumors were surgically removed, weighed and analyzed by
hematoxylin–eosin staining (HE) and IHC. A TUNEL assay was performed on
paraffin-embedded tissue sections according to the manufacturer’s instructions
(Servicebio). The animal study was approved by the Ethics Committee of Southwest
Hospital.

STATISTICAL ANALYSIS

Statistical analyses were performed using SPSS statistical software (version
22.0, Chicago, IL, USA). Data were presented as the mean ± standard deviation of
three independent experiments. Statistical tests for data analysis included
Chi-square test, log-rank test, Student’s t-test, and ANOVA with Turkey’s
method. All tests were two-sided and statistical significance was defined as
p < 0.05. Figures were generated using GraphPad Prism 8 Software.

RESULTS

P-GP, BCRP AND MRP1 PROTEINS WERE OVEREXPRESSED IN GEMCITABINE-RESISTANT
PANCREATIC CANCER TISSUES AND CELLS.

ABC transporter superfamily has seven (A to G) subfamilies with a total of 48
members, and is a special class of proteins in the cell membrane [31]. Studies
have shown that ABC transporters play an important role in gemcitabine
chemotherapy resistance in PC [31, 32]. Among them, P-gp, BCRP and MRP1 are the
recognized molecules that contributed to the development of multidrug resistance
[4, 33]. In this study, IHC staining further confirmed that P-gp, BCRP, MRP1
were overexpressed in gemcitabine-resistant compare with gemcitabine-sensitive
tissues (Fig. 1 A). Western blotting also showed that their expressions in
gemcitabine-resistant PANC-1-GEM cells were higher than that in
gemcitabine-sensitive PANC-1 cells (Fig. 1 B). The expression levels of P-gp,
BCRP, and MRP1 were also markedly upregulated in pancreatic cancer tissues as
compared to the pancreatic non-tumor tissues (Figs. 1I-N). These findings were
validated by data from the Cancer Genome Atlas dataset (TCGA) and the
Genotype-Tissue Expression (GTEx) (Fig. 1O-Q).

Fig. 1

P-gp, BCRP and MRP1 were overexpressed in gemcitabine-resistant pancreatic
cancer tissues and cells. Docking simulation revealed gemcitabine binds to the
substrate binding site of P-gp, BCRP and MRP1 proteins. A IHC staining of P-gp,
BCRP, MRP1 in pancreatic cancer gemcitabine-resistant and gemcitabine-sensitive
tissues, 200x. Scale bar, 50 μm. B Western blotting on the expression of P-gp,
BCRP, MRP1 in the indicated cells. β-actin was used as a loading control.
C-H Interaction between gemcitabine and human P-gp, BCRP and MRP1 protein
models. Details of the best-scoring pose of gemcitabine in the drug binding
pocket of P-gp (C), BCRP (E) and MRP1 (G) binding pocket. 2D diagram of the
interaction between gemcitabine and P-gp (D), BCRP (F) and MRP1 (H) binding
pocket. The cavity of 20 Å was selected as the docking active region, and the
docking calculation was carried out with standard parameters. Purple arrows
represent hydrogen bonding, and green dash lines represent pi-pi interactions.
I-N IHC staining demonstrated the P-gp (I, L), BCRP (J, M) and MRP1 (K, N)
overexpression in PC tissues compared to the pancreatic non-tumor tissues.
O-Q The mRNAs expression profile showed P-gp (O), BCRP (P) and MRP1 (Q)
overexpression in pancreatic cancer tissue as compared to non-tumor tissue
obtained from the public database (TCGA and GTEx). Data are presented as
mean ± SD; *P < 0.05. IHC: immunohistochemistry

Full size image

GEMCITABINE BINDS TO THE SUBSTRATE BINDING SITE OF P-GP, BCRP AND MRP1 PROTEINS

Molecular docking simulation was performed to explore whether gemcitabine
interacted with P-gp, BCRP and MRP1 proteins. The docking analysis showed that
gemcitabine binds to these ABC transporter proteins. The affinity scores of
interactions between gemcitabine and P-gp, BCRP and MRP1 are -8.568, -9.402 and
-10.116 kcal/mol, respectively, indicating that gemcitabine has strong binding
affinity with P-gp, BCRP and MRP1. It was found that the hydroxyl group of
gemcitabine acts as the hydrogen bond receptor with the amino acid residue
Tyr949, while the carbonyl group acts as the hydrogen bond receptor with the
amino acid residue Tyr952 and forms the hydrogen bond interaction with P-gp.
Moreover, the amino acid residues Leu64, Met67, Met68, Met948, Phe982 and Met985
on the binding domain of P-gp were hydrophobic with gemcitabine, which enhanced
the interaction between gemcitabine and P-gp. Gemcitabine formed hydrogen bonds
with amino acid residues Phe432, Thr435 and Asn436 of BCRP, and formed π-π
interaction with Phe439. Amino acid residues Phe431, Phe439, Val546 and Met549
on the BCRP binding domain were hydrophobic with gemcitabine, which enhanced the
interaction between gemcitabine and BCRP. Gemcitabine forms hydrogen bonds with
amino acid residues Lys332, Tyr384, Thr439 and Gln450 of MRP1 receptor. Also,
the amino acid residues Met339, Leu381, Phe385, Tyr440, Met443, Phe594, Pro595,
Ile598 and Trp1245 on the MRP1 binding domain were hydrophobic with gemcitabine,
which enhanced the interaction between gemcitabine and MRP1 (Fig. 1 C-H). These
results suggested that gemcitabine possessed high binding affinity with P-gp,
BCRP and MRP1.

OVEREXPRESSION OF HSA-MIR-3178 PROMOTED GEMCITABINE RESISTANCE IN PANC-1 CELLS
BOTH IN VITRO AND IN VIVO

Our previous study has shown that hsa-miR-3178 overexpression in PC is
associated with poor prognosis [16]. The role of hsa-miR-3178 in gemcitabine
resistance of PC was investigated. qRT-PCR analysis demonstrated the
upregulation of hsa-miR-3178 in seven pancreatic cancer cell lines (AsPC-1,
BxPC-3, CFPAC-1, Hs766t, PANC-1, MIA PaCa2 and SW1990) and in the PC tissues in
contrast to the primary normal human pancreatic duct epithelial cell (HPDE6-C7)
or adjacent non-tumor tissues (Figs. 1 A-C). We further found that hsa-miR-3178
was overexpressed in gemcitabine-resistant PANC-1-GEM cells compared with
gemcitabine-sensitive PANC-1 cells (Fig. 2 D). As shown in Fig. 2 E,
hsa-miR-3178 mimics increased half-inhibitory concentration (IC50) of
gemcitabine (8.06 ± 0.81 μmol/L) compared to control groups (un-transfected
cells, 1.49 ± 0.62 μmol/L; negative control, 1.70 ± 0.18 μmol/L) in PANC-1
cells.

Fig. 2

Over-expression of hsa-miR-3178 promoted proliferation of PANC-1 cells and
conferred gemcitabine resistance. A-C qRT-PCR analysis on hsa-miR-3178
expression in seven pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1,
Hs766t, PANC-1, MIA PaCa2 and SW1990) and in the PC tissues compared to the
primary normal human pancreatic duct epithelial cell (HPDE6-C7) or adjacent
non-tumor tissues. Data are presented as mean ± SD; *P < 0.05, **P < 0.01,
***P < 0.001. D qRT-PCR assay for relative expression of hsa-miR-3178 in PANC-1
and PANC-1-GEM cells. E CCK-8 method was used to detect IC50 of Mock, MNC and
hsa-miR-3178 mimic-transfected PANC-1 cells upon treatment with gemcitabine.
F The CCK-8 growth curves of indicated cells upon treatment with gemcitabine
(1 μmol/L). G The representative fluorescent micrograph and quantification on
EdU staining of indicated cells upon treatment with gemcitabine (1 μmol/L).
H Flow cytometry on cell apoptosis in indicated cells exposed to gemcitabine
(1 μmol/L) for 72 h. I Tumor xenograft images from mice treated with gemcitabine
(5 mg/kg, IP, q3d). J Tumor weights showed the effects of hsa-miR-3178 on the
indicated groups. K Tumor volumes measured on the indicated times. Data are
expressed as mean ± SD from three independent experiments. *P < 0.05;
**P < 0.01; ***P < 0.001. PANC-1-GEM: gemcitabine resistant subline; IC50:
half-inhibitory concentration; Mock: untreated cells; MNC: Mimic negative
control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate; IHC:
immunohistochemistry; IP: intraperitoneal injection

Full size image

The CCK-8 and EdU assays were performed to evaluate cell viability and
proliferation. It was found that PANC-1 cells with induced hsa-miR-3178
expression showed significant resistance to gemcitabine (1 μmol/L), as
manifested by increased cell proliferation (Fig. 2 F, G). We further performed
flow cytometry assay to evaluate whether hsa-miR-3178 was capable of inhibiting
gemcitabine-induced apoptosis in PANC-1 cells. It was found that the percentage
of cell apoptosis in hsa-miR-3178 transfection group was much lower than that in
the control cells (Fig. 2 H).

To further verify the above results, we generated xenograft tumor models via
injecting BALB/c nude mice with PANC-1 cells (with transfection of agomir-3178,
agomir negative control or PBS). As expected, hsa-miR-3178 significantly
enhanced xenograft tumor growth and tumor weight at the end of the experiment
even with gemcitabine treatment (Fig. 2I-K). Furthermore, hsa-miR-3178
overexpression reduced gemcitabine-induced cell apoptosis, and the TUNEL+ cell
proportion decreased compared with the control group (Figure S1 A).

DOWN-REGULATION OF HSA-MIR-3178 INHIBITED PANC-1-GEM CELLS PROLIFERATION AND
INCREASED CELL SENSITIVITY TO GEMCITABINE BOTH IN VITRO AND IN VIVO

To investigate the role of hsa-miR-3178 in in drug resistance in vitro,
PANC-1-GEM cells with hsa-miR-3178 silencing were established by transfecting
hsa-miR-3178 inhibiting vectors. As shown in Fig. 3 A, transfection of
hsa-miR-3178 inhibiting vectors decreased IC50 of gemcitabine
(159.33 ± 0.94 μmol/L) compared to control groups (un-transfected cells,
170.77 ± 1.60 μmol/L; negative control, 170.13 ± 1.30 μmol/L) in PANC-1-GEM
cells. CCK-8 and EdU assays demonstrated that PANC-1-GEM cells with reduced
hsa-miR-3178 expression increased sensitivity to gemcitabine, as manifested by a
significant decrease in cell proliferation (Fig. 3 B, C). Flow cytometry assays
revealed that the percentage of cells apoptosis in hsa-miR-3178 inhibitor
transfection group was significantly higher than that in the control cells which
was the opposite of results of hsa-miR-3178 mimics transfection in PANC-1 cells
(Fig. 3 D).

Fig. 3

Down-regulation of hsa-miR-3178 inhibited proliferation of PANC-1-GEM cells and
increased sensitivity of cells to gemcitabine. A CCK-8 assay on cell viability
of PANC-1-GEM cells transfected with Mock, INC, and hsa-miR-3178 inhibitor and
treated with gemcitabine for calculating IC50. B The CCK-8 growth curves of
indicated cells upon treatment with gemcitabine (150 μmol/L). (C) The
representative fluorescent micrograph and quantification of EdU staining on
indicated cells upon treatment with gemcitabine (150 μmol/L). D Flow cytometry
on cell apoptosis in indicated cells exposed to gemcitabine (150 μmol/L) for
72 h. E Tumor xenograft images from mice treated with gemcitabine (5 mg/kg, IP,
q3d). F Tumor weights showed the effects of hsa-miR-3178 inhibitor on the
indicated groups. G Tumor volumes measured on the indicated times. Data are
expressed as mean ± SD from three independent experiments. *P < 0.05;
**P < 0.01; ***P < 0.001. PANC-1-GEM: gemcitabine resistant subline. IC50:
half-inhibitory concentration; Mock: untreated cells; INC: Inhibitor negative
control; PI: Propidium Iodide; FITC: Fluorescein Isothiocyanate; IP:
intraperitoneal injection

Full size image

To confirm the above results in vivo, we generated xenograft tumor models via
injecting PANC-1-GEM cells (with transfection of antagomir-3178, antagomir
negative control or PBS, respectively) into BALB/c nude mice. As indicated,
antagomir-3178 significantly reduced xenograft tumor weight at the end of the
experiment and slowed down tumor growth following gemcitabine treatment (Fig. 3
E–G). Moreover, hsa-miR-3178 downregulation enhanced the effects of chemotherapy
in vivo, and the TUNEL+ cell proportion increased compared with the control
group (Figure S1 B).

RHOB WAS A DIRECT TARGET GENE OF HSA-MIR-3178

By using four bioinformatics algorithms including miRWalk, miRanda, RNA22 and
Targescan, and then intersecting with down-regulated genes in PANC-1-GEM cells
from gemcitabine resistance dataset GSE80617 [16], we obtained two genes RhoB
and HIST2H2BE. Through literature investigation, we found that RhoB plays a
biological function as a cancer promoter in pancreatic cancer [21]. To explore
the regulatory relationship between RhoB and hsa-miR-3178, we probed the TCGA
database contained RhoB and hsa-miR-3178 expression profiles in PC patients. As
shown in Fig. 4 A, we found that the expression of RhoB was negatively
correlated with hsa-miR-3178.

Fig. 4

RhoB was a direct target gene of hsa-miR-3178. A RhoB was negatively correlated
with hsa-miR-3178 expression in TCGA PC patients. B Western blotting on the
expression of RhoB on PANC-1 cells with hsa-miR-3178 mimics and PANC-1-GEM cells
with hsa-miR-3178 inhibitor, and the expression of RhoB in PANC-1, PANC-1-GEM
cells. C Illustration of putative binding sites of hsa-miR-3178 with the 3’-UTR
of RhoB. D Luciferase assay showed hsa-miR-3178 targeted the 3’-UTR regions of
RhoB. E–F IHC staining of RhoB and Kaplan–Meier analysis (n = 87; Log-rank test;
P = 0.0037). Data are expressed as mean ± SD from three independent experiments.
**P < 0.01; ***P < 0.001

Full size image

Moreover, hsa-miR-3178 mimics was introduced into PANC-1 cells and hsa-miR-3178
inhibitor was introduced into PANC-1-GEM cells. Western blotting revealed that
upregulation of hsa-miR-3178 inhibited RhoB expression while downregulation of
hsa-miR-3178 enhanced RhoB expression (Fig. 4 B).

A dual-luciferase reporter assay was performed to validate RhoB as a direct
target gene of hsa-miR-3178. To do this, hsa-miR-3178 mimics, mimics NC, and
plasmids containing the wild-type 3’-UTR and mutant 3’-UTR were each
constructed, and the characteristics of these vectors are presented in Fig. 4 C
and supplementary-word1. The results indicated that co-transfection of
hsa-miR-3178 mimics and wild-type plasmids significantly decreased luciferase
activity compared with that with the transfection of mutant plasmids (Fig. 4 D).
These results suggested that hsa-miR-3178 may restrain RhoB expression by
directly targeting its 3’-UTR.

RhoB was reported as a cancer suppressor and loss of RhoB was strongly
associated with poor survival of patients [34]. However, the effect of RhoB on
survival in PC has not been studied. We performed IHC assay to explore the
clinical significance of RhoB expression using a tissue microarray which
contains 87 cases of PC. Kaplan–Meier survival analysis revealed that
overexpression of RhoB was significantly associated with a good overall survival
of PC patients (Fig. 4 E–F).

RHOB REVERSED HSA-MIR-3178-MEDIATED GEMCITABINE RESISTANCE IN PC CELLS
GEMCITABINE-RESISTANT PC CELLS AND THE PARENTAL PANC-1 CELLS

To further confirm the role of RhoB suppression by hsa-miR-3178 in PC cell
proliferation and gemcitabine resistance, we co-transfected RhoB overexpression
lentivirus with hsa-miR-3178 mimics in PANC-1 cells or RhoB small interfering
RNA (siRNA) with hsa-miR-3178 inhibitor in PANC-1-GEM cells. As shown in Fig. 5
A, upregulation of RhoB decreased the IC50 of PANC-1 cells to gemcitabine
(Vector + MNC, 1.68 ± 0.16 μmol/L; RhoB + MNC, 0.39 ± 0.02 μmol/L) and reversed
hsa-miR-3178-mediated gemcitabine resistance in PANC-1 cells (Vector + Mimics,
6.87 ± 0.30 μmol/L; RhoB + Mimics, 1.44 ± 0.10 μmol/L). On the contrary,
downregulation of RhoB increased the IC50 of PANC-1-GEM cells to gemcitabine
(Scr + INC, 168.93 ± 0.75 μmol/L; siRhoB + INC, 190.07 ± 0.60 μmol/L) and
antagonized hsa-miR-3178 inhibitor-mediated gemcitabine re-sensitization in
PANC-1-GEM cells (Scr + Inhibitor, 155.40 ± 0.93 μmol/L; siRhoB + Inhibitor,
169.10 ± 1.07 μmol/L) (Fig. 6 A). Furthermore, CCK-8 and EdU assay results
revealed that RhoB overexpression reduced proliferation of PANC-1 cells and
reversed the promotion of cell proliferation by hsa-miR-3178 in PANC-1 cells
(Fig. 5 B, C). However, RhoB knockdown induced proliferation of PANC-1-GEM cells
and antagonized the inhibition of cell proliferation by hsa-miR-3178 inhibitor
in PANC-1-GEM cells (Fig. 6 B, C).

Fig. 5

RhoB reversed hsa-miR-3178-mediated proliferation and gemcitabine resistance in
PANC-1 cells. Co-transfection of RhoB overexpression lentivirus with
hsa-miR-3178 mimics in PANC-1 cells. A CCK-8 assay for IC50 of gemcitabine in
PANC-1 cells with indicated transfection. B The CCK-8 growth curves of indicated
cells upon treatment with gemcitabine (1 μmol/L). C The representative
fluorescent micrograph and quantification of the EdU staining of indicated cells
upon treatment with gemcitabine (1 μmol/L). D Flow cytometry on cell apoptosis
in indicated cells exposed to gemcitabine (1 μmol/L) for 72 h. Data are
expressed as mean ± SD from three independent experiments. *P < 0.05;
**P < 0.01; ***P < 0.001. IC50: half-inhibitory concentration; Mock: untreated
cells; MNC: Mimic negative control; vector: vector-only control; PI: Propidium
Iodide; FITC: Fluorescein Isothiocyanate

Full size image
Fig. 6

RhoB reversed hsa-miR-3178-mediated proliferation and gemcitabine resistance in
PANC-1-GEM cells. Co-transfection of siRhoB with hsa-miR-3178 inhibitor in
PANC-1-GEM cells. A CCK-8 assay for IC50 of gemcitabine in PANC-1-GEM cells with
indicated transfection. B The CCK-8 growth curves of indicated cells upon
treatment with gemcitabine (150 μmol/L). C The representative fluorescent
micrograph and quantification of the EdU staining of indicated cells upon
treatment with gemcitabine (150 μmol/L). D Flow cytometry on cell apoptosis in
indicated cells exposed to gemcitabine (150 μmol/L) for 72 h. Data are expressed
as mean ± SD from three independent experiments. *P < 0.05; **P < 0.01;
***P < 0.001. IC50: half-inhibitory concentration; Mock: untreated cells; INC:
Inhibitor negative control; vector: vector-only control; siRhoB: RhoB small
interfering RNA; Scr: scramble control; PI: Propidium Iodide; FITC: Fluorescein
Isothiocyanate

Full size image

In addition, flow cytometric analysis revealed that overexpression of RhoB
promoted apoptosis of PANC-1 cells and also antagonized the suppression of cell
apoptosis by hsa-miR-3178 in PANC-1 cells (Fig. 5 D). On the contrary, RhoB
knockdown suppressed cell apoptotic rate compared with control group and
reversed the increased apoptosis by hsa-miR-3178 inhibitor in PANC-1-GEM cells
(Fig. 6 D). Taken together, these results demonstrated that RhoB could reverse
hsa-miR-3178-mediated proliferation and gemcitabine resistance in PC cells.

HSA-MIR-3178/RHOB AXIS REGULATED GEMCITABINE RESISTANCE BY PI3K/AKT SIGNALING
PATHWAY AND ABC TRANSPORTERS

Previous studies have reported that RhoB negatively regulates the PI3K/Akt
signaling pathway [20, 35]. As shown in figure S1 A-B, IHC assay revealed that
hsa-miR-3178 promoted expression of p-PI3K and p-Akt 473 in vivo. To explore the
association of hsa-miR-3178, RhoB and PI3K/Akt signaling pathway, we performed
Western blotting analyses to probe the protein expression involved in PI3K/Akt
signaling pathway in PANC-1 cells and PANC-1-GEM cells with indicated
treatments.

As shown in Fig. 7 A, RhoB overexpression decreased expression of p-PI3K, p-Akt
308 and p-Akt 473, while total PI3K and total Akt expression was not affected in
PANC-1 cells. Co-transfection of RhoB and hsa-miR-3178 mimics reduced the
expression of p-PI3K, p-Akt 308 and p-Akt 473 by hsa-miR-3178 in PANC-1 cells.
On the contrary, RhoB knockdown increased the expression of p-PI3K, p-Akt 308
and p-Akt 473 and co-transfection of siRhoB and hsa-miR-3178 inhibitor reversed
the downregulated expression of p-Akt 308 and p-Akt 473 by hsa-miR-3178
inhibitor in PANC-1-GEM cells (Fig. 7 B).

Fig. 7

Hsa-miR-3178/RhoB axis mediated tumor growth and gemcitabine resistance by
PI3K/Akt signaling pathway in PC cells. A-F Western blotting on the expression
of RhoB, phosphorylated-PI3K, total PI3K, phosphorylated-Akt 308,
phosphorylated-Akt 473, total Akt and P-gp, BCRP, MRP1 in the indicated cells.
β-actin was used as a loading control. IC50: half-inhibitory concentration

Full size image

To further confirm the role of PI3K/Akt signaling pathway involved in regulating
proliferation and gemcitabine resistance via hsa-miR-3178/RhoB axis in PC,
PANC-1 cells and PANC-1-GEM cells were incubated with the PI3K inhibitor
LY294002 and PI3K agonist 740Y-P. As expected, the stimulatory effect of
hsa-miR-3178 overexpression on p-PI3K, p-Akt 308 and p-Akt 473 was inhibited by
PI3K inhibitor in PANC-1 cells (Fig. 7 C). On the contrary, the inhibition of
downregulated hsa-miR-3178 on p-PI3K, p-Akt 308 and p-Akt 473 was reversed by
PI3K agonist in PANC-1-GEM cells (Fig. 7 D). In addition, Hsa-miR-3178
overexpression in PANC-1 cells increased expression of P-gp, BCRP and MRP1 in
vitro and in vivo (Fig. 7 E, Figure S1 A), whereas, hsa-miR-3178 inhibitor
decreased the expression of P-gp, BCRP and MRP1 in vitro and in vivo (Fig. 7 F,
Figure S1 B). To explore the regulatory relationship between PI3K/Akt signaling
pathway and ABC transporters, we probed the TCGA and GTEx data contained PIK3CA,
PIK3CB, PIK3CD, PIK3CG, AKT1 and the three ABC transporters expression profiles.
As shown in Figure S2, we found that the expression of PI3K/Akt was positively
correlated with the ABC transporters. These results suggested that hsa-miR-3178
induces gemcitabine resistance by activating PI3K/Akt signaling pathway-mediated
ABC transporters in PC cells (Fig. 8).

Fig. 8

Schematic illustration of potential mechanisms of hsa-miR-3178/RhoB axis
mediates tumor growth and gemcitabine resistance via PI3K/Akt pathway-mediated
overexpression of ABC transporters in PC cells

Full size image

DISCUSSION

The mechanism by which ABC transporters regulate gemcitabine resistance in
pancreatic cancer remains unclear. In this study, we propose that hsa-miR-3178
inhibits RhoB to promote gemcitabine resistance in pancreatic cancer via
activating PI3K/Akt signaling pathway and upregulating ABC transporters. We
found that P-gp, BCRP and MRP1 were highly expressed in gemcitabine-resistant
pancreatic cancer tissues and cells by IHC and Western blotting. Molecular
docking assay demonstrated that gemcitabine had high binding affinity with P-gp,
BCRP and MRP1. Previous studies suggested that miRNAs have an important
regulatory relationship with ABC transporters [36]. Our previous study indicates
that hsa-miR-3178 is associated with gemcitabine resistance in pancreatic cancer
[16]. However, the mechanism of miR-3178 in regulating ABC transporter to induce
gemcitabine resistance is not clear. In the present study, we identified
overexpression of hsa-miR-3178 in gemcitabine-resistant PANC-1-GEM cells as
compared to its parental PANC-1 cells. Gain- and loss-of-function studies
revealed that hsa-miR-3178 induces cellular proliferation and gemcitabine
resistance in PC cells. By bioinformatics analysis, Western blotting, and dual
luciferase reporter assay, we predicted and confirmed that hsa-miR-3178 was a
potential upstream miRNA of RhoB. And IHC assay and Kaplan–Meier survival
analysis indicated that higher RhoB level was significantly associated with
better overall survival of PC patients. We further investigated the important
role of hsa-miR-3178/RhoB axis in regulating cellular gemcitabine resistance via
PI3K/Akt signaling pathway. In vitro cell experiments and in vivo animal
experiments confirmed that hsa-miR-3178 overexpression could upregulate the
expression of PI3K/Akt and P-gp, BCRP and MRP1. A number of studies have
confirmed that PI3K/Akt signaling pathway is related to chemotherapy resistance
caused by ABC transporters [37, 38]. Our results suggested that increased
hsa-miR-3178 activates PI3K/Akt signaling pathway, upregulates the expression of
ABC transporters and aggravates gemcitabine resistance in pancreatic cancer.
Thus, hsa-miR-3178 could be a potentially therapeutic target for PC treatment.

Previous studies have reported the roles of miRNAs on gemcitabine-based
chemotherapy in various cancers [39]. In breast cancer, low expression of
miR-205-5p leads to upregulation of ERp29 and decreased sensitivity of cancer
cells to gemcitabine [40]. In PC, by delivering miR-210, exosomes derived from
gemcitabine-resistant cancer stem cells reinforce gemcitabine resistance [41].
Our previous research found that hsa-miR-3178 was associated with gemcitabine
resistance in pancreatic cancer [13]. In this study, we identified its mechanism
in promoting gemcitabine resistance.

As a well-known tumor suppressor, RhoB was reported to be relevant to cisplatin
resistance [18, 22, 42]. Also, previous studies have reported that RhoB was
targeted by miRNAs [20, 43]. In this study, we demonstrated that hsa-miR-3178
promoted gemcitabine resistance of PC by targeting RhoB, and upregulation of
RhoB could reverse hsa-miR-3178-mediated gemcitabine resistance. The regulatory
networks of RhoB are very complex. Particularly, it was reported that RhoB
negatively regulates the PI3K/Akt signaling pathway. In lung cancer, loss of
RhoB accelerates lung cancer progression through activation of PI3K/Akt
signaling pathway [35]. In osteosarcoma, miR-19 targets RhoB and down-regulates
its expression, inhibiting the dephosphorylation of Akt1 protein and promoting
tumor cell metastasis [20]. In line with these reports, our results demonstrated
that RhoB overexpression leads to overexpression of p-PI3K, p-Akt 308 and p-Akt
473 in PANC-1 cells; whereas RhoB knockdown leads to lower their expression in
PANC-1-GEM cells. The results of co-transfection with RhoB and hsa-miR-3178 and
treatment with PI3K inhibitor LY294002 and PI3K agonist 740Y-P further suggested
that RhoB reduces proliferation and gemcitabine resistance by antagonizing the
PI3K/Akt signaling pathway in PC cells.

Research has found that overexpression of ABC drug transporters, such as P-gp,
BCRP and MRP1 confers an acquired MDR due to their capabilities of transporting
a broad range of chemically diverse anticancer drugs. For example, P-gp and MRP1
induced gemcitabine resistance in pancreatic cancer cells [4, 31,32,33]. Our
previous study uncovered that PI3K inhibitor BAY-1082439 was able to reverse
P-gp-mediated drug resistance in oral epidermoid carcinoma cell line and
non-small cell lung cancer cell line, and the mechanism of action of BAY-1082439
is to inhibit PI3K which can downregulate the expression of P-gp and BCRP [37].
Since P-gp plays an important role in MDR, it has become an urgent task for
researchers to find effective P-gp inhibitors [44, 45]. However, no positive
results were obtained in clinical trials on P-gp inhibitors due to drug toxicity
caused by the non-specific distribution of inhibitors and the pharmacokinetic
changes caused by the interaction with a variety of anti-tumor drugs [46]. The
limitation of this study is that only one resistant cell line was used for
validation. Subsequent gemcitabine-resistant cell lines in pancreatic cancer
need further validation of our conclusions. Another limitation is we cannot
implement the biochemical experiments to prove the binding of ABC transporters
to gemcitabine. Further validation was need to be performed.

CONCLUSION

In conclusion, this study indicates that the hsa-miR-3178/RhoB/PI3K/Akt-mediated
upregulation of ABC transporters play a vital role in inducing gemcitabine
resistance in pancreatic cancer cells. Targeting hsa-miR-3178/RhoB may represent
a potential therapeutic strategy to circumvent ABC transporters-mediated
gemcitabine resistance for PC treatment.

AVAILABILITY OF DATA AND MATERIALS

Not applicable.

ABBREVIATIONS

PC:

Pancreatic Cancer

miRNAs:

MicroRNAs

RhoB:

Ras Homolog Family Member B

PANC-1-GEM:

Gemcitabine-resistant PANC-1 cell

ABC transporters:

ATP-Binding Cassette Transporters

P-gp:

P-glycoprotein

BCRP:

Breast Cancer Resistance Protein

MRP1:

Multidrug Resistance Protein 1

INC:

Inhibitor Negative Control

MNC:

Mimic Negative Control

Scr:

Scramble Control

IHC:

Immunohistochemistry

CCK-8:

Cell Counting Kit 8

EdU:

5-Ethynyl-2’-deoxyuridine

HE:

Hematoxylin–Eosin Staining

TUNEL:

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling

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ACKNOWLEDGEMENTS

We would like to thank Professor Dong-Hua Yang from College of Pharmacy and
Health Sciences, St. John's University for her valuable comments on the
manuscript. We wished to sincerely thank Chuanming Xie, Yujun Zhang, Jiejuan
Lai, Li Shuai of the Institute of Hepatopancreatobiliary Surgery, Southwest
Hospital, Army Medical University in Chongqing for technical support of animal
experiments in this study.

FUNDING

This work was supported by the Science and Technology Project of Guangzhou
(202206010093, to Yingfang Fan) and the Key-Area Research and Development
Program of Guangdong Province, China (2020B010165004, to Yingfang Fan) and the
National Key R&D Program of China (No. 2017YFC1308600, to Huaizhi Wang) and the
National Natural Science Foundation of China (No. 81672382, to Huaizhi Wang).

AUTHOR INFORMATION

Author notes

1. Jianyou Gu and Wenjie Huang contributed equally to this work.

AUTHORS AND AFFILIATIONS

1. Department of Hepatobiliary Surgery I, Zhujiang Hospital, Southern Medical
University, Guangzhou, Guangdong, People’s Republic of China

Jianyou Gu, Wenjie Huang, Tian Tao, Jinsui Li & Yingfang Fan

2. Institute of Hepatopancreatobiliary Surgery, Chongqing General Hospital,
Chongqing, People’s Republic of China

Jianyou Gu, Wenjie Huang, Xianxing Wang, Junfeng Zhang, Yao Zheng, Songsong
Liu, Jiali Yang & Huaizhi Wang

3. Department of Pharmaceutical Sciences, College of Pharmacy and Health
Sciences, St. John’s University, Queens, NY, 11439, USA

Zhe-Sheng Chen & Chao-Yun Cai

Authors
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CONTRIBUTIONS

Jianyou Gu and Wenjie Huang contributed to the conception, design, acquisition
of data and data analysis, obtaining the findings, and drafting the manuscript.
Xianxing Wang, Junfeng Zhang, Tian Tao, Yao Zheng, Songsong Liu, Jiali Yang,
Zhe-Sheng Chen, Chao-yun Cai and Jinsui Li contributed to acquisition of data
and technical support. Yingfang Fan and Huaizhi Wang offered conception, design
and critical revision of the manuscript for important intellectual content. All
authors have contributed to the completion and endorsement of the final
manuscript. The authors read and approved the final manuscript.

CORRESPONDING AUTHORS

Correspondence to Huaizhi Wang or Yingfang Fan.

ETHICS DECLARATIONS

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

The animal study and the use of the clinical specimens were approved by the
Ethics Committee of Southwest Hospital.

CONSENT FOR PUBLICATION

Not applicable.

COMPETING INTERESTS

The authors declare no competing interests.

ADDITIONAL INFORMATION

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SUPPLEMENTARY INFORMATION

ADDITIONAL FILE 1: SUPPLEMENTARY WORD 1.

h-RHOB 3-UTR reporter vector.

ADDITIONAL FILE 2: SUPPLEMENTARY FIGURE 1.

Hsa-miR-3178 restrainsexpression of RhoB and promotes expression of
phosphorylated-PI3K andphosphorylated-Akt 473 in vivo. (A, B) IHC on
theexpression of RhoB, phosphorylated-PI3K, phosphorylated-Akt 473, P-gp, BCRP
andMRP1 in the indicated groups. And the TUNEL+ cell proportion in the
indicatedgroups. Scale bar, 50 μm.Data are expressed as mean ±SD from three
independentexperiments. **P < 0.01; ***P < 0.001. IHC: immunohistochemistry.
TUNEL:Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling.

ADDITIONAL FILE 3: SUPPLEMENTARY FIGURE 2.

PI3K/Aktwere positively correlated with the three ABC transporters in TCGA and
GTExdatabases.

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Gu, J., Huang, W., Wang, X. et al. Hsa-miR-3178/RhoB/PI3K/Akt, a novel signaling
pathway regulates ABC transporters to reverse gemcitabine resistance in
pancreatic cancer. Mol Cancer 21, 112 (2022).
https://doi.org/10.1186/s12943-022-01587-9

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* Received: 30 December 2021

* Accepted: 27 April 2022

* Published: 10 May 2022

* DOI: https://doi.org/10.1186/s12943-022-01587-9

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KEYWORDS

* Hsa-miR-3178
* RhoB
* Gemcitabine resistance
* PI3K/Akt
* Pancreatic cancer
* ABC transporters

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* Conclusion
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