Background
Pancreatic ductal adenocarcinoma (PDAC) is the most deadly malignancy, with a overall survival rate of 7% 5 years after diagnosis [
1]. The poor prognosis is due in part to the lack of diagnostic symptoms during the early stages of this disease, and approximately 80%~ 85% of patients with PDAC are not candidates for resection at the time of diagnosis. Chemotherapy, mostly gemcitabine and gemcitabine-based combinations, is commonly used to treat of these unresectable PDAC patients. However, owing to the intrinsic or external factors that promote chemotherapy resistance, among the gemcitabine-treated patients with metastatic disease, the 1-year survival rate is only 17 to 23% [
2]. Among patients with metastatic PDAC, 34.5% of these patients present with disease progression during gemcitabine treatment [
3]. Thus, a better understanding and quicker ability to recognize mechanisms of gemcitabine chemoresistance may provide new treatments and better drug selection strategies for improving the prognosis of this lethal disease.
MicroRNAs (miRNAs) are a class of small non-coding RNAs that are composed of 19~ 25 nucleotides. They negatively regulate genes at the post-transcriptional level by binding to the 3′ or 5′ untranslated region (UTR) of target mRNAs. In recent years, many studies have revealed that miRNA dysregulation is involved in PDAC carcinogenesis and drug resistance by serving as oncogenes or tumor suppressors [
4]. Indeed, recent evidence has shown that dysregulated miRNAs participate in PDAC pathogenesis, including cell proliferation, apoptosis, differentiation, invasion, migration, epithelial-mesenchymal transition (EMT), angiogenesis, etc. In addition, several studies have demonstrated that some miRNAs are critical contributors to drug resistance by regulating the above crucial processes and are important determinants of anticancer therapy efficacy in PDAC. Thus, a better understanding of the biology and underlying mechanisms of drug resistance-related miRNAs may provide potential therapeutic targets for improving PDAC prognosis.
Previously, miR-10a-5p has been reported to be overexpressed and to act as an important mediator of metastasis formation in PDAC [
5,
6]. However, the essential role and underlying mechanism of miR-10a-5p in PDAC chemoresistance remain unclear. In our previous study, we identified a panel of dysregulated miRNAs associated with drug resistance in PDAC via miRNA microarray analysis of the established gemcitabine-resistant PDAC cell line AsPC-1-Gem (data unpublished). Among these miRNAs, miR-10a-5p had a high expression level (≥ 5-fold change). Thus, we presumed that miR-10a-5p might be involved in drug resistance development in PDAC. We then found that miR-10a-5p was up-regulated in the gemcitabine-resistant PDAC cells AsPC-1-Gem and enhanced PDAC cells resistance to gemcitabine in vitro and vivo. In addition, we also found that miR-10a-5p promoted the migratory and invasive ability of PDAC cells by activating the EMT signaling pathway. Next, we confirmed that TFAP2C is a direct target gene of miR-10a-5p. Consistently, TFAP2C overexpression resensitized PDAC cells to gemcitabine, which was triggered by miR-10a-5p. Further studies revealed that TFAP2C also decreased the migration and invasion capability of PDAC cells. Finally, survival analysis revealed that high miR-10a-5p expression levels and low TFAP2C expression levels were both independent adverse prognostic indicators in patients with PDAC. Therefore, these results together indicate that miR-10a-5p/TFAP2C may be new therapeutic targets and prognostic markers in PDAC.
Methods
Cell lines, culture and transfection
The human pancreatic ductal adenocarcinoma (PDAC) cell lines AsPC-1, BxPC-3, MiaPaCa-2, PANC-1, Su86.86 and T3M4 cells were donated by Dr. Freiss H. (University of Heidelberg, Heidelberg, Germany). The 293A cell line was purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College (IBMS, CAMS/PUMC). The AsPC-1, BxPC-3 and Su86.86 cell lines were maintained in RPMI 1640 (HyClone Logan, UT, USA). The 293A, MiaPaCa-2, PANC-1 and T3M4 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Logan, UT, USA). All media were supplemented with 10% fetal bovine serum (FBS, HyClone) at 37 °C with 5% CO2. AsPC-1-Gem cells are gemcitabine-resistant AsPC-1 cells and were obtained by researchers in our lab gradually increasing the gemcitabine doses. Gemcitabine (750 ng/ml) was added into the medium to maintain the resistant AsPC-1-Gem cell phenotype for a long time. Gemcitabine was removed 1 month before the cells were used experimentally.
T3M4, Su86.86 and AsPC-1 cells were chosen for further studies (for details, see the Results), and were transfected with 50-100 nM oligonucleotides with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) in 6-well plates (5 × 10
5 cells/well). All the steps were carried out according to the protocol provided by the Lipofectamine 2000 manufacturer. The miR-10a-5p mimics, miR-10a-5p inhibitor and matched controls were synthesized by Genepharma (Shanghai, China). All the oligonucleotides used are shown in Table
1.
Table 1
Correlations of miR-10a-5p and TFAP2C levels in tissues and clinicopathological parameters
Gender | | | 0.270 | | | 0.670 |
Male | 24 | 30 | | 28 | 27 | |
Female | 11 | 25 | | 16 | 19 | |
Age(years old) | | | 0.380 | | | 0.831 |
<65 | 23 | 30 | | 25 | 28 | |
≥ 65 | 12 | 25 | | 19 | 18 | |
Locations | | | 0.532 | | | 0.372 |
Head | 23 | 37 | | 27 | 33 | |
Body-tail | 12 | 18 | | 17 | 13 | |
Perineuronal invasion | | | 1.000 | | | 0.019 |
No | 20 | 31 | | 19 | 32 | |
Yes | 15 | 24 | | 25 | 14 | |
Tumor staging | | | 0.790 | | | 0.426 |
T1/T2 | 29 | 44 | | 34 | 39 | |
T3/T4 | 6 | 11 | | 10 | 7 | |
Lymph node staging | | | 0.519 | | | 0.832 |
N0 | 23 | 31 | | 27 | 27 | |
N1 | 12 | 24 | | 17 | 19 | |
TNM staging | | | 0.084 | | | 0.505 |
I | 21 | 22 | | 22 | 21 | |
II | 14 | 33 | | 22 | 25 | |
Diabetes | | | 0.399 | | | 0.169 |
No | 27 | 47 | | 39 | 35 | |
Yes | 8 | 8 | | 5 | 11 | |
RNA extraction and real-time PCR (qRT–PCR)
PDAC cells were transfected into 6-well plates (5 × 10
5 cells/well) for 48 h. Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA). The RNA quality was evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, USA) at 260- and 280-nm (A260/280) wavelengths. Complementary DNA was synthesized by TaqMan MicroRNA RT Kit (Applied Biosystems) and the reverse transcription kit (Promega, Madison, WI). Quantitative RT–PCR (qRT-PCR) was performed using TaqMan MicroRNA Assays (Applied Biosystems) and SYBR Green Master Mix (Takara, Japan). U6 RNA and GAPDH were chosen as internal controls for mRNA and miRNA detection, respectively. The relative expression of miR-10a-5p and mRNAs was calculated through the 2
-ΔΔCT method. All the primers used are shown in Table
1.
Cell proliferation and growth inhibition assay
At 24 h after transfection, PDAC cells were plated into 96-well culture plates (1000 cells/well) for cell proliferation assays. All the plates were cultured at 37 °C with 5% CO2. For cell proliferation assays, 10 μL/well cell count kit (CCK-8) reagent was added at 0, 24, 48 and 72 h after plating. After an additional 2.5-h incubation with CCK-8 reagent at 37 °C, the optical density (OD) at the 450-nm wavelength (OD450) was measured using a microplate reader (Wellscan MK3, Thermo/Labsystems, Finland). OD630 served as a reference, and the OD in the blank well was used as the base level. For growth inhibition assays, 4000 cells/well were plated into 96-well culture plates at 24 h after transfection. After incubation for 4-6 h for cell adherence, a gemcitabine (Eli Lilly and Company) concentration gradient from 100 nM to 1 mM or control PBS buffer was added into each well. Cell count kit (CCK-8) reagent (10 μL/well) was added after an additional 48-h incubation at 37 °C. Then, the inhibition rate was calculated as follows: OD
sample
= OD450 − OD630, \( \mathrm{Inhibition}\ \mathrm{rate}=1-\frac{OD_{\mathrm{Gem}}-{OD}_{\mathrm{blank}}}{OD_{\mathrm{PBS}}-{OD}_{\mathrm{blank}}} \).
Apoptosis assay
PDAC cells were transfected into 6-well plates and treated with 10 μM gemcitabine 24 h later. After treating for 48 h, the cells were collected and resuspended in binding buffer. Next, the cells were stained with Annexin V-FITC and propidium iodide (PI) (Beyotime, China) according to the manufacturer’s instructions. The analysis was carried out using flow cytometry (FACScan; BD Biosciences, USA).
In vitro transwell assays
Transwell assays were performed in triplicate using transwell migration chambers (8-μm pore size; Corning, USA) for cell migration and invasion experiments. For invasion assays, wells were coated with diluted ECM solution (Sigma-Aldrich, Shanghai, China). Cells transfected with miR-10a-5p mimics, inhibitors or paired control oligonucleotides were transferred to the top of the upper chambers or to the ECM gel in the serum-free culture. After culturing for 48 h, medium containing 10% FBS was added to the lower chambers. The cells that migrated or invaded into the lower surface after 48 h of incubation were fixed in 90% ethyl alcohol and stained with hematoxylin-eosin for counting. The number of cells in the chamber were counted in five random visual fields under a microscope at 100× magnification. The average number of cells counted in the five fields was used as the final result. All experiments were performed three times.
Western blotting
After transfection for 48 h, total cellular protein was extracted with RIPA lysis buffer (Applygen, Beijing). Total protein (100 μg) was separated on SDS-PAGE gel and then transferred to a PVDF membrane (Millipore, Billerica, MA). The membrane was probed with primary antibodies (1:1000, Danvers, MA) overnight at 4 °C after blocking with 5% non-fat milk at room temperature for 1 hour. The next day, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody (1:3000, Applygen, Beijing) at room temperature for 1 hour. Protein bands were detected by ECL reagents (Millipore, Billerica, MA).
Animal experiments
AsPC-1 cells stably transfected with miR-10a-5p lentiviral vectors or control (Lv-AsPC-1-miR-10a-5p or Lv-AsPC-1-NC) were injected subcutaneously into the right flank of 6-week-old female BALB/c mice (Shanghai, Chinese Academy of Sciences, China) (5 × 106 cells in 250 μl of PBS per mouse). Each experimental group included five mice. Gemcitabine (50 mg/kg) was administered by intraperitoneal injections 1 week after tumor formation (tumor size between 100 to 200 mm3), followed by periodic booster shots every 3 days for 4 weeks. Two perpendicular tumor diameters was measured once a week using a caliper. Tumor volume (mm3) was calculated: volume (mm3) = 1/2 × length × width2. All tumor-bearing mice were euthanized on the 35th day.
Fluorescent reporter assay
The pmirGLO dual luciferase miRNA target expression vector (Promega, E1330) was used to assess miR-10a-5p regulation of miRNA target sites. Wild-type (WT) or mutant (MUT) miR-10a-5p binding site sequences in the 3’-UTR of TFAP2C were synthesized by Invitrogen and cloned into pmirGLO vectors at the 3′-end of the firefly luciferase gene. Vectors and miR-10a-5p mimics or mimics controls were co-transfected into 293A cells with Lipofectamine 2000 reagent in 12-well plates. Forty-eight hours later, luciferase activity was evaluated using a Dual-Luciferase Reporter Assay system (Promega). Renilla luciferase (hRlucneo) served as the control reporter for normalization.
Patients and sample collection
Formalin-fixed, paraffin-embedded PDAC tissues (n = 90) and matched tumor-adjacent tissues (n = 90) were collected from PUMCH and were made tissue microarrays. None of the patients received neoadjuvant therapy before surgical resection. PDAC was diagnosed and staged by pathological examination by two pathologists independently. Patients with TNM stages later than stage II were not included in this research. Follow-up data were obtained from medical records and follow-up. The end-point was overall survival (OS). Survival time was defined according to the time between the date of death or the last follow-up date and the operation date.
Pancreatic tissue collection and in situ hybridization (ISH)
The miRCURY LNA detection probe (18017-15; Exiqon, Vedbaek, Denmark) was used to detect the miR-10a-5p expression levels in tissue microarrays. After deparaffinization in xylene and rehydration with graded alcohol washes, the slides were fixed in 4% paraformaldehyde for 20 min. After washing in PBS three times, the slides were incubated with 15 μg/ml proteinase K for 15 min at room temperature. The slides were then washed in PBS and fixed in 4% paraformaldehyde for 15 min. After rinsing in PBS, the slides were prehybridized in hybridization buffer for 1 h at 50 °C and then hybridized in probe-containing hybridization buffer overnight at 4 °C. The following day, the slides were washed stringently at 50 °C for 20 min, followed by blocking in a blocking solution for 1 h at room temperature. Finally, the slides were placed in blocking solution containing alkaline phosphatase-conjugated anti-DIG Fab fragment overnight at 4 °C again. The colorimetric detection reaction was carried out using the NBT/BCIP kit (Thermo Fisher Scientific). The slides were scored according to the staining intensity and percentage of positive cells. The scoring for stain intensity was as follows: none (0 points), weak staining (1 point), intermediate staining (2 points), and strong staining (3 points). The scoring for the positive cell percentage was as follows: absent (0 points), 1–24% positive cells (1 point), 25–49% (2 points), 50–74% (3 points), and 75–100% (4 points). The final score was calculated by multiplying the above two scores. MiR-10a-5p expression was considered to be low if the final score was less than the median value and high if the final score was the median value or above.
Immunohistochemistry (IHC)
Rabbit anti-human TFAP2C polyclonal antibodies (ab218107, Abcam) were used for staining. Slides were deparaffinized in xylene and rehydrated in a graded alcohol series. After washed with PBS, endogenous peroxidase activity of the slides was blocked with 3% H2O2 for 10 min. Incubate the slides in 0.1% trypsin for antigen retrieval and heating them in a microwave oven. The slides were incubated with primary antibody (1:200) overnight at 4 °C then. After washed three times with PBS, the slides were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at 37 °C. Diaminobenzidine served as a chromogen. The slides were then counterstained with hematoxylin. Nonimmune rabbit serum served as the negative control. TFAP2C expression levels were scored according to the staining intensity and percentage of positive cells, either. The scoring for stain intensity was as follows: none (0 points), weak staining (1 point), intermediate staining (2 points) and strong staining (3 points). The scoring for the positive cell percentage was as follows: absent (0 points), 1–24% of the cells (1 point), 25–49% of the cells (2 points), 50–74% of the cells (3 points), and 75–100% of the cells (4 points). The final score was calculated by multiplying the above two scores. TFAP2C expression was considered to be low if the final score was less than the median value and was considered high if the final score was the median value or above.
Sequence of the primers used in this study.
Oligonucleotides/Primer | Sequence (5′–3′) |
MiR-10a-5p mimics sense | CAAAUUCGGAUCUACAGGGUAUU |
MiR-10a-5p mimics antisense | UACCCUGUAGAUCCGAAUUUGUG |
Mimics control sense | UUCUCCGAACGUGUCACGUTT |
Mimics control antisense | UACCCUGUAGAUCCGAAUUUGUG |
MiR-10a-5p inhibitor | CACAAAUUCGGAUCUACAGGGUA |
Inhibitor control | CAGUACUUUUGUGUAGUACAA |
TFAP2C sense | ATCTTGGAGGACGAAATGAGAT |
TFAP2C antisense | CAGATGGCCTGGCTGCCAA |
GAPDH sense | CGGAGTCAACGGATTTGGTCGTAT |
GAPDN antisense | AGCCTTCTCCATGGTGGTGAAGAC |
Discussion
Chemoresistance is one of the main causes of poor prognosis in PDAC. Thus, investigating the mechanisms underlying chemoresistance and chemotherapy resensitization in PDAC cells is critical for PDAC treatment. In the present study, we identified that miR-10a-5p was up-regulated in gemcitabine-resistant PDAC cells and found that miR-10a-5p enhanced PDAC cell resistance to gemcitabine in vitro and vivo. In addition, miR-10a-5p promoted the migratory and invasive ability of PDAC cells though up-regulating EMT-related gene expression. Mechanistically, miR-10a-5p directly targeted TFAP2C to confer gemcitabine resistance. Meanwhile, TFAP2C acted as a tumor suppressor to decrease the PDAC cell migration and invasion capability and negatively modulated EMT-associated gene expression. We also demonstrated that high miR-10a-5p expression and low TFAP2C expression are significantly associated with poor prognosis in patients with PDAC. In this regard, our data indicated that miR-10a-5p/TFAP2C were valuable prognostic predictors of PDAC and appeared to be promising targets for PDAC therapy.
It has been reported that miR-10a-5p plays varying but important roles in multiple cancers. Wang et al. [
7] found that miR-10a-5p suppresses the miR-10a-EphA4 axis, promoting cell proliferation, invasion and EMT in hepatic cell cancer. In non-small cell lung cancer (NSCLC), in vitro experiments revealed that miR-10a-5p overexpression promoted NSCLC cell proliferation, migration and invasion by directly targeting PTEN [
8]. In breast cancer [
9], miR-10a-5p promotes cell migration, which is positively regulated by RUNX2. In cervical cancer [
10], miR-10a-5p promotes cell colony formation, migration and invasion by targeting CHL1. However, in other studies, miR-10a-5p acts very differently. In gastric cancer, miR-10a-5p represses cell growth, migration and invasion through silencing HoxA1 [
11]. In breast cancer [
12], one article reported that miR-10a-5p was significantly down-regulated in malignant cells compared with normal or benign glandular cells, indicating that miR-10a-5p might act as a tumor suppressor. Regarding tumor chemosensitivity, miR-10a-5p also plays controversial roles. Studies have shown that miR-10a-5p is associated with cisplatin (DDP) resistance in lung cancer. Silencing miR-10a-5p in DDP-resistant cells increases cell chemosensitivity to DDP, induces cell apoptosis and up-regulates caspase 3/8 expression and activity [
13]. However, in ER-positive breast cancer [
14], Cox regression analysis revealed that increased miR-10a-5p expression is associated with a long relapse-free time following tamoxifen treatment. Our study was the first to investigate the differential miR-10a-5p expression in gemcitabine-resistant and parental cell lines. We found that miR-10a-5p was significantly up-regulated in gemcitabine-resistant cells and promoted PDAC cell migration and invasion in vitro. Further studies revealed that miR-10a-5p enhances gemcitabine resistance in vitro and vivo.
MiR-10a-5p has also been reported to be overexpressed in cancer cells compared with normal tissues [
10,
15‐
17] and to be up-regulated in metastatic [
16] or recurrent [
9] tumor cells compared with primary cancer cells. Li et al. [
18] identified a relationship between the miR-10a-5p level and both disease-free survival and OS in gastric cancer. In NSCLC [
8], miR-10a-5p expression is higher in highly metastatic cells rather than in weakly metastatic cells or normal tissues, as determined by miRNA expression microarray, and relative factor analysis reveals that high miR-10a-5p expression is associated with later lymph node (N) and metastasis (M) stages. Our study used ISH to reveal that miR-10a-5p is up-regulated in PDAC tissue samples compared with matched tumor-adjacent tissues. We found no correlation between miR-10a-5p levels and clinicopathological parameters. However, univariate and multivariate survival analysis both indicated that miR-10a-5p expression (high) is an independent adverse prognostic factor in PDAC.
Recent studies on TFAP2C have mainly focused on breast cancer and lung cancer. In breast cancer, TFAP2C participates in regulating luminal-specific genes [
19] and is involved in multiple cell proliferation pathways, indicating that TFAP2C is a potential drug target site [
20]. TFAP2C may also be associated with breast cancer prognosis: higher TFAP2C levels correlate with poor overall survival in unique ERα(+) and endocrine therapy-treated subgroups [
21], and high tissue TFAP2C expression also contributes to the failure of anti-hormone treatments [
22]. In NSCLC, Kang, J. et al [
23] found that TFAP2C overexpression is associated with cell tumorigenesis and cell cycle activation via the miR-183 and miR-33a pathways in vivo. Other researchers have suggested that the slug-miR-137-TFAP2C axis may provide new candidate target molecules for lung cancer therapeutics [
24]. However, in our study results, TFAP2C works as a tumor suppressor and can suppress PDAC cell migration and invasion, as well as negatively regulate certain protein levels. On the other hand, silencing TFAP2C up-regulates p21 levels. Furthermore, univariate and multivariate analysis both demonstrated that low TFAP2C expression was an independent adverse prognostic factor. Our study is the first to investigate the roles of TFAP2C in PDAC and to elucidate the tumor suppresser role of this protein.