Journal of Cancer 2019, Vol.

10 5031

Ivyspring
International Publisher
Journal of Cancer
2019; 10(21): 5031-5040. doi: 10.7150/jca.31191
Research Paper

Down-regulation of miR-30a-5p is Associated with Poor

Prognosis and Promotes Chemoresistance of
Gemcitabine in Pancreatic Ductal Adenocarcinoma
Liangjing Zhou, Shengnan Jia, Guoping Ding, Mingjie Zhang, Weihua Yu, Zhengrong Wu, Liping Cao
Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, No. 3, Qingchun Road, Hangzhou, Zhejiang province,
China

 Corresponding author: Liping Cao, MD, Department of General Surgery, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou
310000, China; Tel/Fax: (011) 86-0571-86090073; [email protected]

© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.

Received: 2018.11.03; Accepted: 2019.08.06; Published: 2019.08.28

Abstract
MicroRNA-30a-5p (miR-30a-5p) plays an important role in many biological and pathological
processes, and therefore has been studied extensively. However, its expression and function in
pancreatic ductal adenocarcinoma (PDAC) remain unclear. Furthermore, whether miR-30a-5p
affects sensitivity of PDAC cells to gemcitabine (GEM) is worthy of further exploration. The results
showed that miR-30a-5p expression in pancreatic cancer was decreased and the down-regulated
expression correlated with poor prognosis, while up-regulating miR-30a-5p suppressed tumor cell
proliferation, cell cycle and increased apoptosis. MiRNA expression profiles between
gemcitabine-resistant pancreatic cancer cells and parental pancreatic cancer cells showed significant
change of miR-30a-5p expression. Besides, up-regulating miR-30a-5p in PDAC significantly increased
the chemosensitivity of gemcitabine. Furthermore, FOXD1 is a direct target of miR-30a-5p and the
miR-30a-5p/FOXD1/ERK axis may play an important role in the development of gemcitabine
resistance in pancreatic cancer. In summary, our study showed that miR-30a-5p increases the
sensitivity of pancreatic cancer to gemcitabine, and it may be a potential therapeutic target to
overcome gemcitabine resistance.
Key words: miR-30a-5p, FOXD1, prognosis biomarker, pancreatic cancer, gemcitabine, sensitive

Introduction
Pancreatic cancer is a common, highly malignant reported that intracellular drug metabolism-related
neoplasm with a 5-year survival rate of only 3%–6% 1. genes (such as hENT1, hCNT1) and apoptosis-related
It is the fourth leading cause of cancer-related deaths genes (such as IAP families and HMGA1) may affect
in the United States. Chemotherapy of pancreatic the therapeutic efficacy of gemcitabine 4-7. However,
cancer can remarkably improve survival and quality the exact mechanism of such resistance is still unclear.
of life for patients with resectable or unresectable More gene related to the chemosensitivity of
pancreatic cancer 2. In 1997, Burris et al demonstrated gemcitabine needs to explore.
that gemcitabine significantly improved symptoms MiRNAs have been discovered in recent years.
and prolonged median survival in a phase III trial 3. They are small, non-coding RNAs with a length of 19–
Currently, gemcitabine has become the preferred 22 nucleotides. These molecules are considered as key
chemotherapeutic drug for advanced pancreatic regulatory factors for the occurrence and
cancer. However, recent studies have found that a development of tumors. Because miR-30a-5p plays an
large proportion of patients are resistant to important role in many biological and pathological
gemcitabine, leading to treatment failure. It was processes, it has attracted much attention 8. Li et al

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found that miR-30a-5p inhibits breast cancer cell Tissue samples

proliferation, migration, and invasion by regulating Formalin-fixed and paraffin-embedded
the ERK/Ets-1 signaling pathway 9. In a study by pancreatic ductal adenocarcinoma and adjacent
Kumarswamy et al, miR-30a-5p affects the normal pancreatic tissue specimens were obtained
epithelial-mesenchymal transition of cells by acting from pancreatic cancer patient. The research protocol
on the target gene Snail, thereby inhibiting the was reviewed and approved by the Research Ethics
invasion and metastasis of lung cancer 10. However, Committee of Sir Run Run Shaw Hospital, School of
its expression and mechanism in pancreatic cancer Medicine, Zhejiang University. The study was
remain unclear. By searching the Gene Expression conducted following the Helsinki Declaration. All
Omnibus (GEO) database, we found that expression participants or their guardians gave written consent
of miR-30a-5p was low in pancreatic cancer, and its of their tissue samples and medical information to be
function worth further exploring. used for scientific research.
Recent studies indicate that miR-30a-5p is also
involved in resistance of chemotherapeutic drugs. Yu Fluorescence in situ hybridization (FISH)
et al demonstrated that miR-30a-5p acts as an For the detection of miR-30a-5p expression in
autophagy inhibitor in chemotherapy of chronic tumor sections, fluorescence in situ hybridization
myeloid leukemia, which promotes the assay was performed using the following LNA oligos
chemotherapy-induced cytotoxic response by sequences: LNA-miR-30a-5p 5’-
inhibiting the expression of autophagy genes BECNL UGUAAACAUCCUCGACUGGAAG-3’, which were
and ATG5 11. However, it is unclear whether constructed by GenePharma Corporation (Shanghai,
miR-30a-5p is involved in gemcitabine resistance of China). Briefly, the formalin-fixed paraffin-embedded
pancreatic cancer. We found that expression of tissue sections were deparaffinized in xylene and
miR-30a-5p was significantly down-regulated in rehydrated in serial ethanol solutions. The slides were
gemcitabine-resistant pancreatic cancer cells. Thus, in then treated with proteinase K for 20 min at 37°C.
the present study we focused on the effect and Slides were prehybridized in prehybridization buffer
mechanism of miR-30a-5p in promoting the at the 78°C for 8 min. After prehybridization, slides
chemosensitivity of gemcitabine in PDAC. were hybridized in hybridization buffer with specific
Cy3-labeled probes at 37°C for 16 h. The sections were
Materials and Methods washed with SSC thoroughly to remove the
background signals. Nuclei were counterstained with
Cell culture
DAPI, and then sections were analyzed and imaged
Human pancreatic cancer cell lines, including using a fluorescence microscope (Olympus, Japan).
Panc-1, BxPC-3, MIAPaCa-2 and normal pancreatic
ductal epithelial cell HPDE6-C7 were all purchased Transient transfection
from Chinese Academy of Sciences (Shanghai, China) MiR-30a-5p mimics (miR10000087-1-5), FOXD1
and were cytogenetically tested and authenticated siRNA (stQ0001131-1) and their matched negative
before they were frozen. BxPC-3 cell line was cultured control (NC) were synthesized by Guangzhou
in RPMI 1640 medium containing 10% fetal bovine RiboBio Co., Ltd. (Guangzhou, China). Recombinant
serum (FBS, Gibco, New York, USA). Panc-1, plasmids over-expressing FOXD1 and matched
MIAPaCa-2 and HPDE6-C7 cell lines were cultured in negative control were constructed by Shanghai
DMEM medium with 10% FBS. For drug treatment, Genechem Co.,Ltd (GOCP2301022229). Briefly, 1×105
cells were plated into 6-well plates, and 0.4 ug/ml cells were seeded onto 6-well culture plates. Cell lines
gemcitabine (Eli Lilly and Company. USA) was added were transfected in serum-free medium, transfection
to the culture. Cells were collected or tested 48 h after was performed using Lipofectamine 3000 following
the drug treatment. the manufacturer’s protocol (Invitrogen). After 24 h of
transfection, cells were kept in a culture medium
RNA extraction and qRT-PCR
containing 10% FBS up to 48 h. The cells were then
Total RNA of cells was isolated and purified by harvested and followed by PCR or Western blot
miRNeasy Mini Kit (Qiagen, Maryland, USA), analysis.
following the manufacturer’s instructions. Reverse
transcription (RT) was performed using PrimeScript Cell proliferation assay
RT reagent Kit (Takara, Otsu, Japan) following the Cell proliferation was measured via CCK-8
manufacturer’s instructions. The polymerase chain assay. Cells were seeded at a density of 5×103/well
reaction (PCR) primers of miR-30a-5p and U6 used into 96-well plates and cultured for 72 h. The cells
were purchased from Guangzhou RiboBio Co., Ltd. were then incubated with 20 μl CCK-8 for 2 h at 37˚C.
(MQP-0101 and MQP-0202,Guangzhou, China). The absorbance at 490 nm was recorded.

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Journal of Cancer 2019, Vol. 10 5033

Cell apoptosis assays transfection. Renilla luciferase activity was used for
For cell apoptosis analysis, cells were collected normalization, and firefly luciferase activity was
by trypsinization and washed three times using PBS, detected with a dual luciferase reporter assay kit
and then cells were treated with AnnexinV-PE and according to the manufacturer’s protocol.
7AAD for 30 min according to the instruction manual. Statistical Analysis
Samples were acquired on a BD FACSAria III Cell
All data were presented as mean ± standard
Sorting System (Becton Dickinson, New York, USA)
deviation (SD) and analyzed using student’s t-test. p
before analysis using the BD FACSDiva software 6.1.3
value < 0.05 was considered as statistically
(Becton Dickinson).
significance. All data were processed using SPSS,
Cell cycle analysis version 19.0 and Graphpad Prism 5.0 software
Cells were harvested and washed with PBS, program.
fixed with 75% ethanol overnight at 4℃. The nuclei of
the cells were stained with propidium iodide (PI) for
Results
30 min and analyzed with a fluorescence-activated Low miR-30a-5p expression in pancreatic
cell sorting caliber system. The results were presented cancer cell lines and tissues
as the percentages of cells in each phase.
By analyzing GSE24279 and GSE29352 in the
Western blot GEO database, we found that miR-30a-5p expression
in pancreatic cancer tissues was lower than those in
Cells were harvested and total protein lysates
normal paraneoplastic tissues (Figure 1A and 1B,
were resolved using 10% sodium dodecyl sulfate–
p<0.05). Also, the expression level of miR-30a-5p in
polyacrylamide gels and electroblotted onto to a
pancreatic cancer cell lines BxPC-3, MIAPaCa-2 and
polyvinylidene fluoride membrane, blocked by 5%
Panc-1 was verified lower compared with the normal
skim milk, and probed with the following antibodies:
pancreatic duct epithelial cell line HPDE6-C7
FOXD1 (1:1000, ab49156, Abcam), ERK (1:1000, 4695,
(p<0.05). Among them, the fold change was most
Cell Signaling Technology), p-ERK (1:1000, 4370, Cell
significant in BxPC-3 cells (Figure 1C, p<0.05).
Signaling Technology) and GAPDH (1:5000,
Furthermore, 60 cases of pancreatic cancer
ab181602, Abcam). The membrane was then
tissues were collected, and miR-30a-5p expression
incubated with the secondary anti-rabbit secondary
was detected by fluorescence in situ hybridization.
antibody and visualized by enhanced
MiR-30a-5p was mainly located in cytoplasm, and the
chemiluminescence using Kodak X-OMAT LS film
expression of miR-30a-5p in the pancreatic cancer
(Eastman Kodak, Rochester, USA).
tissues was lower than those in normal pancreatic
Animal experiments tissues (Figure 1D). Patients were classified into two
PDAC-bearing male nude mice with groups according to miR-30a-5p expression, and we
subcutaneous passage of BxPC-3 were used. When the assessed the potential correlation of miR-30a-5p
tumor size reached approximately 5 mm in length, the expression with clinicopathological features and
mice were divided into two groups (3 mice per group) prognosis. We found that decreased expression of
randomly. Stabilized miRNAs (miR-30a-5p agomir miR-30a-5p tended to be poorly differentiated (Table
and Negative control agomir) were purchased from 1, p<0.05). No significant differences were observed
RiboBio (miR40000087-1-10, Guangzhou, China). according to other clinicopathological features. We
MiR-30a-5p agomir (5 nM) or control oligos mixture also followed up the patients and found that the
was injected into the xenografts in a multi-site decreased expression of miR-30a-5p showed a
injection manner every 3 days for two weeks, and significantly lower survival rate than the high
then followed by administration with gemcitabine expression group (Figure 1E, p<0.05).
(100 mg/kg). At the end of the 30-day observation MiR-30a-5p overexpression inhibits cell
period, mice were killed and the tumors were proliferation, cell cycle and promotes
measured. The tumor volume was measured with a apoptosis
caliper, using the formula volume = length ×
width2/2. To determine the function of miR-30a-5p in
pancreatic cancer, we used miR-30a-5p mimics to
Luciferase assay induce miR-30a-5p overexpression in cell line BxPC-3
The cells were co-transfected with pLMP vectors and Panc-1 (Figure 2A, p<0.05). The cell counting kit-8
containing FOXD1 3'UTR and miR-30a-5p mimics. assay showed that the cell proliferation rate was
Cells were harvested and subjected to lysis 48 h after decreased significantly after miR-30a-5p
overexpression (Figure 2B, p<0.05). The apoptosis

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level was increased after miR-30a-5p overexpression Parameters miR-30a-5p expression P values
Low High
(Figure 2C, p<0.05). Moreover, flow cytometry results ≤4cm 21 20 0.781
indicated that the percentage of cells at G0/G1 phase >4cm 9 10
in tumor cells with miR-30a-5p mimics was T stage
T1-2 7 4 0.317
significantly increased and the proportion at S and T3-4 23 26
G2/M phase was decreased (Figure 2D, p<0.05). Lymph node metastasis
Based on these findings, miR-30a-5p overexpression No 9 10 0.781
Yes 21 20
inhibits progression in pancreatic cancer. Distant metastasis
M0 28 28 1.000
M1 2 2
Table 1: Relationship between miR-30a-5p expression and TNM stage
clinicopathological features I-II 10 7 0.486
III -IV 20 21
Parameters miR-30a-5p expression P values
* P<0.05
Low High
Age
<60 11 7 0.26
≥60 19 23 MiR-30a-5p increases the sensitivity of
Gender pancreatic cancer to gemcitabine
Male 15 16 0.796
Female 15 14 By analyzing GSE80616 in the GEO database, the
Smoking history differentially expressed miRNAs in gemcitabine-
Yes 23 25 0.519
resistant pancreatic cancer cell line were screens out
No 7 5
Tumor site (Figure 3A), and we discovered that the miR-30a-5p
Head 22 25 0.347 expression was significantly decreased (Figure 3B,
Body + Tail 8 5
Pathological grade
p<0.05).
Poor and Middle 25 18 0.045*
High 5 12
Tumor size

Figure 1. Low miR-30a-5p expression in pancreatic cancer cell lines and tissues. A and B. Relative miR-30a-5p expression level in pancreatic cancer tissues and adjacent normal
tissues in a public data set (GSE24279 and GSE29352). C. The expression level of miR-30a-5p in pancreatic cancer cell lines with the normal pancreatic duct epithelial cell line
HPDE6-C7 as control. D. The expression level of miR-30a-5p in pancreatic cancer tissue was detected by FISH. E. Effect of the miR-30a-5p expression level on overall survival
in pancreatic cancer patients. Data are expressed as mean ± SEM (n = 3). *indicated p<0.05

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Figure 2. MiR-30a-5p overexpression inhibits cell proliferation, cell cycle and promotes apoptosis. A. MiR-30a-5p was verified over-expressed in BxPC-3 and Panc-1 cell lines
by the transfection of miR-30a-5p mimics. B. The CCK-8 assay showed cell proliferation rate decreased significantly after miR-30a-5p overexpression. C. The flow cytometry
assay showed the apoptosis level increased significantly after miR-30a-5p overexpression. D. Flow cytometry assay showed the percentage of cells at G0/G1 phase was increased
after miR-30a-5p overexpression. Data are expressed as mean ± SEM (n = 3). *indicated p<0.05

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 Journal of Cancer 2019, Vol. 10 5036

Based on the results, we hypothesized that the xenograft tumor model of PDAC on nude mice.
change of miR-30a-5p expression may affect the MiR-30a-5p expression was confirmed significantly
chemosensitivity of gemcitabine in pancreatic cancer increased in subcutaneous tumors after intratumoral
cells. Pancreatic cancer cells were treated with injection of miR-30a-5p agomir by FISH (Figure 3D). It
gemcitabine following miR-30a-5p overexpression, was observed that the miR-30a-5p over-expressed
and the killing effect of gemcitabine was increased tumors were obviously smaller than the control ones
significantly (Figure 3C, p<0.05). To further confirm after they were under the equal dose of gemcitabine
the role of miR-30a-5p in vivo, we established treatment (Figure 3E, p<0.05).

Figure 3. MiR-30a-5p increases the sensitivity of pancreatic cancer to gemcitabine. A. Analysis of the change of miRNAs expression in gemcitabine-resistant pancreatic cancer
cell line based on the GEO databases GSE80616. B. Expression of miR-30a-5p in gemcitabine-resistant pancreatic cancer cell line was decreased compared with parental cell line.
C. The killing effect of gemcitabine increased in miR-30a-5p over-expressed cells compared to negative control. D. The miR-30a-5p was confirmed increased in the subcutaneous
tumors after the intratumoral injection of miR-30a-5p agomir. E. MiR-30a-5p agomir or control oligos mixture (n=3 in each group) was injected into the xenografts in a multi-site
injection manner every 3 days for two weeks, and then followed by administration with gemcitabine. Mice were sacrificed at the end of the 30-day observation period and the
tumor volume was measured. Data are expressed as mean ± SEM (n = 3). *indicated p<0.05

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Figure 4. FOXD1 is a direct target for miR-30a-5p. A. The 3’ UTR of FOXD1 contains a complementary matching region of miR-30a-5p through predictive analysis by
bioinformatics websites. B. The expression level of FOXD1 in pancreatic cancer cell lines with the normal pancreatic duct epithelial cell line HPDE6-C7 as control. C. Relative
FOXD1 expression level in pancreatic cancer tissues and adjacent normal tissues in a public data set (GSE16515). D. The expression level of FOXD1 in pancreatic cancer tissue
was detected by IHC. E. Expression of FOXD1 in cell lines transfected with miR-30a-5p mimics was detected by qRT-PCR. F. Expression of FOXD1 in cell lines transfected with
miR-30a-5p mimics was detected by western blot analysis. G. Luciferase activity of the construct containing the FOXD1 3’-UTR reporter gene or mutant type (MT) FOXD1 3'
UTR in cell lines co-transfected with the miR-30a-5p mimics. Relative Renilla luciferase activity was determined and normalized against firefly luciferase activity. Data are
expressed as mean ± SEM (n = 3). *indicated p<0.05

(Figure 4D). To confirm specific regulation of FOXD1

FOXD1 is a direct target for miR-30a-5p by miR-30a-5p, we carried out a transfection
We used bioinformatics websites TargetScan and experiment. Compared with control cells,
miRDB for predictive analysis. The 3'-UTR of FOXD1 miR-30a-5p-overexpressing cells had significantly
contains a complementary region to miR-30a-5p decreased FOXD1 mRNA and protein levels (Figure
(Figure 4A). The FOXD1 expression in pancreatic 4E and 4F, p<0.05). To further assess whether FOXD1
cancer cell lines was verified much higher compared is a direct target of miR-30a-5p, we performed
with the normal pancreatic duct epithelial cell line luciferase reporter assays. MiR-30a-5p overexpression
HPDE6-C7 (Figure 4B). By analyzing GSE16515 in the significantly decreased activity of the 3′-UTR of the
GEO database, FOXD1 expression in pancreatic luciferase reporter gene. However, miR-30a-5p
cancer tissues was higher than those in normal exhibited no effect on the pLMP reporters containing
paraneoplastic tissues (Figure 4C, p<0.05). Also mutant type FOXD1 3' UTR (Figure 4G, p<0.05),
FOXD1 expression was detected by IHC and the confirming that miR-30a-5p directly targeted FOXD1
expression of FOXD1 in the pancreatic cancer tissues mRNA.
was stronger than those in normal pancreatic tissues

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Overexpression of miR-30a-5p increases the the up-regulated FOXD1 inhibited the effect of
sensitivity of pancreatic cancer to gemcitabine miR-30a-5p in promoting the sensitivity of
by targeting FOXD1. gemcitabine (Figure 5D). Previous studies showed
By analyzing the mRNA changes of the previous that activation of FOXD1 may induce the ERK
gemcitabine-resistant cell line based on GSE80617 signaling pathway and chemotherapeutic drug
(Figure 5A), it showed that FOXD1 expression was resistance 12,13, and we also confirmed that ERK
significantly increased (Figure 5B). Based on the direct signaling pathway was inhibited after FOXD1
interaction between miR-30a-5p and FOXD1, we knockdown in pancreatic cancer (Figure 5E).
hypothesized whether miR-30a-5p affected the Furthermore, in line with the results in
chemosensitivity of gemcitabine via FOXD1. FOXD1-knockdown cells, we found that FOXD1 and
Pancreatic cancer cells were treated with gemcitabine p-ERK expression were significantly lower in the
following FOXD1 knockdown, and the killing effect of miR-30a-5p overexpressing cells compared with
gemcitabine increased significantly (Figure 5C, controls (Figure 5F). Therefore, the overexpression of
p<0.05). Furthermore, when simultaneously miR-30a-5p may increase the sensitivity of pancreatic
over-expressing miR-30a-5p and FOXD1, it showed cancer to gemcitabine by targeting FOXD1.

Figure 5. Overexpression of miR-30a-5p increases the sensitivity of pancreatic cancer to gemcitabine by targeting FOXD1. A. Analysis of the change of mRNAs expression in
gemcitabine-resistant pancreatic cancer cell line based on the GEO databases GSE80617. B. Expression of FOXD1 in gemcitabine-resistant pancreatic cancer cell line was
increased compared with parental cell line. C. The killing effect of gemcitabine increased in FOXD1 knockdown cells compared to negative control. D. FOXD1 overexpression
inhibited the effect of miR-30a-5p on increasing the sensitivity of gemcitabine. E. The expression of ERK signaling was detected in FOXD1 knockdown or control cells. F. The
expression levels of FOXD1 and ERK were analyzed by western blot in cell lines treated with miR-30a-5p mimics or negative control. Data are expressed as mean ± SEM (n =
3). *indicated p<0.05

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miR-30a-5p/FOXD1 axis might be a therapeutic

Discussion strategy for pancreatic cancer.
Recent studies have shown that miRNAs are In summary, the present study showed that
closely related to the development of tumors. expression of miR-30a-5p was low in pancreatic
Microarray analyses indicate that miR-30a-5p cancer, and overexpression of miR-30a-5p inhibited
expression decreases in breast, gastric, and rectal cell proliferation, cell cycle and promoted apoptosis.
cancer, suggesting that miR-30a-5p may be a tumor In addition, study showed that overexpression of
suppressor gene 14,15. However, the expression and miR-30a-5p increased chemosensitivity of pancreatic
function of miR-30a-5p in pancreatic cancer have not cancer cells to gemcitabine. FOXD1 is a direct target of
been reported. In this study, miR-30a-5p expression miR-30a-5p, and the miR-30a-5p/FOXD1/ERK axis
was also low in pancreatic cancer cell lines and tumor played an important role in the development of
tissues, and over-expressing miR-30a-5p significantly gemcitabine resistance in pancreatic cancer. In future
inhibited the cell proliferation, cell cycle and tailored therapies, miR-30a-5p may be a potential
promoted apoptosis. Thus, miR-30a-5p may be a therapeutic target to overcome gemcitabine resistance
potential therapeutic target. in pancreatic cancer patients.
Recent studies also have found a correlation
between low expression of miR-30a-5p and resistance Acknowledgments
to chemotherapeutic drugs. It was reported that low This work was supported by the National
miR-30a-5p expression in tumors may promote Natural Science Foundation of China [grant number:
expression of Beclin-1, an autophagy-related protein, 81772548]; Major Research Project of Science
thereby promoting the autophagy level and Technology Department of Zhejiang Province [grant
enhancing the efficacy of cisplatin and adriamycin number: 2015C03G2010160]; Zhejiang provincial
16.17. In addition, Meng et al showed that miR-30a-5p
health and Family Planning Commission project
overexpression increases the chemotherapeutic effect [grant number: 2015KYB218 and 2018KY102].
of EGFR inhibitor on non-small cell lung cancer by
modulating the PI3K/AKT signaling pathway 18. Competing Interests
However, no study investigated the role of The authors have declared that no competing
miR-30a-5p in gemcitabine resistance. Among studies interest exists.
on gemcitabine resistance in pancreatic cancer, it has
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