Background
Pancreatic cancer, one of the most aggressive malignancies, ranks the fourth cause of cancer-related death. Currently, there is lack of sensitive screening for early stage tumors. Up to date, surgery is the effective treatment for pancreatic cancer, however, the 5-year survival rate of the patients with pancreatic cancer is lower than 10% [
1]. Unfortunately, 80% of the patients are not suitable for surgery and the recurrence rate in those who subjected to resection is very high [
2]. Chemotherapy remains the primary treatment and the combination therapy including FOLFIRINOX, paclitaxel and gemcitabine is the first-line drug, but the outcome is not satisfactory [
3,
4]. In terms of targeted drugs, the tyrosine-kinase inhibitor of epidermal growth factor receptor, Eroltinib, combined with gemcitabine, just made modest progress in survival benefit [
5]. Consequently, the mechanisms underlying pancreatic cancer should be further investigated.
Recently, emerging evidence indicated the crucial roles of non-coding RNAs in various human diseases, and non-coding RNAs can be sub-grouped to small non-coding RNAs (< 200 nucleotides) and long non-coding RNAs (lncRNAs; > 200 nucleotides) [
6]. LncRNAs take part in various physiological and pathological processes, including proliferation, differentiation, invasion, or chromosome inactivation [
7]. Studies showed that lncRNA H19 was overexpressed in human pancreatic ductal adenocarcinoma and induced cancer cell proliferation by modulating cell cycle through E2F1 pathway [
8]. LncRNA HOXA-AS2 was also up-regulated in pancreatic cancer tissues. Mechanistically, it promoted pancreatic cancer cell proliferation by interacting with enhancer of zeste homolog 2 and lysine specific demethylase 1 [
9]. In addition, the microRNAs belongs to a class of small non-coding RNA with 18–22 nucleotides in length [
10,
11]. Up to date, hundreds of identified miRNAs have been shown to function as key regulators in tumor development including pancreatic cancer. For example, miR-1271 served as a potent tumor suppressor by reducing AKT/mTOR signaling and promoting apoptosis [
12]. Overexpression of miR-126 and miR-34a had superior anti-tumor effect in pancreatic cancer [
13]. The lncRNA DLX6 antisense RNA 1 (DLX6-AS1) is a developmentally-regulated long non-coding RNA. According to GTEx Analysis, it is overexpressed in brain tissues in normal. High DLX6-AS1 expression was noticed in lung adenocarcinoma and associated with histological differentiation and TNM stage [
14]. DLX6-AS1 was also up-regulated in hepatocellular carcinoma tissue and correlated with clinical prognosis [
15]. However, how DLX6-AS1 regulates pancreatic cancer tumorigenesis and its underlying molecular mechanisms regarding pancreatic cancer development remain unknown.
In the present study, we detected the expression of DLX6-AS1 in pancreatic cancer tissues and cells and predicted its potentially targeted miRNA. We demonstrated that DLX6-AS1 up-regulated the expression of Zinc finger E-box-binding homeobox 2 (ZEB2) by sponging miR-181b, therefore promoting cell proliferation, invasion as well as epithelial-mesenchymal transition in pancreatic cancer cells.
Materials and methods
Human specimens
Eighty-four pairs of cancerous and adjacent normal tissues were collected from patients diagnosed with pancreatic cancer from 2015 to 2017 at the First People’s Hospital of Changzhou. All the specimens were frozen in liquid nitrogen and stored at − 80 °C for further use. The study was approved by the Ethics Committee of the First People’s Hospital of Changzhou and written informed consent was signed and returned by each patient before using the tissues and clinical data.
Cell culture
Human pancreatic duct epithelial cell Line (HPDE6-C7), and human pancreatic cancer cell lines CAPAN-1, BxPC-3, SW 1990 and PANC-1were purchased from ATCC (Manassas, USA). CAPAN-1 and BxPC-3 cells were cultured in RPMI-1640 medium (Gibco, Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific). PANC-1cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Thermo Fisher Scientific) supplemented with 10% FBS (Gibco Thermo Fisher Scientific). SW 1990 cells were cultured in Leibovitz’s L-15 medium and HPDE6-C7 was maintained in Keratinocyte Serum Free Medium supplemented with 25 mg/500 ml Bovine Pituitary Extract and 2.5 µg/500 ml Epidermal Growth Factor (Gibco, Thermo Fisher Scientific). They were all kept at 37 °C in a humidified atmosphere containing 5% CO2.
Oligonucleotides and transfection
The small interfering RNAs (siRNAs) for DLX6-AS1 [siDLX6-AS1 and siDLX6-AS1(a)] and the respective negative control siRNA, miR-181b mimics (5′-AACAUUCAUUGCUGUCGGUGGGU-3′), miR-181b inhibitors (5′-ACCCACCGACAGCAAUGAAUGUU-3′) and the respective negative control miRNAs were purchased from RiboBio (Guangzhou, China). Cell transfections (final concentration for siRNA transfection: 100 nM, final concentration for miRNAs transfection: 50 nM) were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol and the transfection efficiency was confirmed using quantitative real-time PCR (qRT-PCR). All the experiments were performed in triplicate.
RNA isolation and qRT-PCR
Total RNA was extracted from tissue or cells through TRIzol reagent (Invitrogen, Carlsbad, USA). The extracted RNA was then reversely transcribed into cDNA using PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Dalian, China). Real-time PCR was performed with SYBR Premix Ex Taq (Takara, Dalian, China) in the ABI7500 system. GAPDH or U6 was used as internal control for data analysis. All the experiments were performed in triplicate.
CCK-8 assay
For proliferation assay, the transfected cells were seeded in 96-well plates at the density of 5000 cells/well over night. At 0, 24, 48 and 72 h after transfection, the cells were incubated with 10 µl CCK-8 reagent for 2 h at 37 °C in the dark, and the OD value at 450 nm was read using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) to determine the viability. All the experiments were performed in triplicate.
The transfected cells were seeded in a 6-well plate at the density of 1000 cells/well and incubated in full culture medium at 37 °C. At 14 day, the cells were washed with PBS, fixed with methanol and stained with 1% crystal violet. The number of colonies was counted under a microscope. All the experiments were performed in triplicate.
Transwell migration and invasion assays
Transwell migration and invasion assays were measured by Transwell chamber (8 µm pore size, Corning) without Matrigel for migration assay and with Matrigel for invasion assay. At 48 h after transfection, 1 × 105 cells were cultured in the upper chamber with serum-free median. The lower chamber was filled with median containing 10% FBS as the chemoattractant. After incubation for 48 h, cells in the upper membrane were removed with cotton swab while those that migrated or invaded were fixed in methanol, stained with 0.1% crystal violet and quantified under a microscope. All the experiments were performed in triplicate.
Luciferase reporter assays
DLX6-AS1 fragments with the wild type (WT) or mutant (MUT) miR-181b binding sites were cloned to generate the plasmids pmirGLO-DLX6-AS1-WT or pmirGLO- DLX6-AS1-MUT (Promega, Madison, USA). The 3′UTR of ZEB2 containing the wild type or mutant miR-181b binding sites were used to generate the plasmids pmirGLO-ZEB2-WT and pmirGLO-ZEB2-MUT (Promega). HEK293T cells were co-transfected with luciferase plasmids and miR-181b mimics, or mimics NC by using Lipofectamine 2000 reagent according to the manufacture’s instruction (Invitrogen). At 48 h after transfection, firefly and Renilla luciferase activities were measured by a Dual-Luciferase Reporter Assay System (Promega) and the experiments were performed in triplicate.
Western blot
The tissue or cells were collected and total proteins were extracted and quantified. Equal amount of proteins was subjected to 10% SDS-PAGE and then transferred onto PVDF membranes (Millipore, Bedford, USA). After blocking with 5% fat-free milk, the membranes were probed with primary anti-ZEB2, anti-E-cadherin, anti-vimentin or anti-N-cadherin antibodies (Abcam, Cambridge, USA) at 4 °C overnight. Then membranes were incubated with horseradish peroxidase-conjugated IgG secondary antibody (Santa Cruz Biotechnology, Santa Cruz, USA) at 37 °C for 2 h. Enhanced chemiluminescence kit (Pierce, Waltham, China) with imaging system (Bio-Rad, Hercules, USA) were used to analyze the protein signals and β-actin was used as the internal control.
Lentivirus expressing shDLX6-AS1 or shRNA control were designed and packaged by Genechem (Shanghai, China), and stable cell lines were established by infecting lentivirus into SW1990 cells and selected by puromycin (Sigma, St. Louis, USA). All the animal experiments were approved by the Animal Experimentation Ethics Committee of the First People’s Hospital of Changzhou Hospital. For the tumor growth assay, twelve female BABL/c athymic nude mice (4–5 weeks old) were subjected to flank subcutaneous injection of SW1990 cells stably expressing shDLX6-AS1 or shNC. Tumor volume was measured every 5 day from the 10 day to the 35 day after inoculation. The volume was calculated as: volume = (length × width2)/2. At 35 day, the mice were sacrificed and the tumors were removed, weighed and snap-frozen for RNA extraction. For the tumor metastasis assay, twelve female BABL/c athymic nude mice (4–5 weeks old) were subjected to tail vein injection of SW1990 cells stably expressing shDLX6-AS1 or shNC. At 35 day, all the mice were sacrificed and the lung were excised, and the number of metastatic nodules in the lung was counted.
Statistical analysis
Data were presented as mean ± standard deviation. The data analysis was performed by using GraphPad Prism software (Version 5.0, GraphPad, San Diego, USA). The significant differences in the clinical parameters between low expression and high expression of DLX6-AS1 groups were analyzed by Chi square test. Significant differences for the mean values between groups were determined by the Student’s t test or one-way ANOVA, as appropriate. P value < 0.05 was considered statistically significant.
Discussion
In the early stage of pancreatic cancer, there are almost no symptoms. When patients have typical symptoms, they have already reached an advanced stage and showed tumor metastasis [
16]. As a result, the prognosis for pancreatic cancer is poor and the 5-year overall survival is very low with median survival of only 6 months [
17,
18]. These make us to seek more effective therapies and advanced strategies for the treatment of pancreatic cancer.
Accumulating evidence has demonstrated the critical roles of lncRNAs in regulating pathological processes of pancreatic cancer. For instance, ectopic expression of AB209630 suppressed cell proliferation ability in pancreatic ductal adenocarcinoma cells resistant to gemcitabine through blocking the PI3K/AKT signaling pathway [
19]. UCA1 was up-regulated in pancreatic cancer tissues and correlated with poor clinical outcome, and promoted cell migration and invasion by the Hippo signaling pathway [
20]. XLOC_000647 served as a tumor suppressor, impairing cell proliferation, invasion, and epithelial-mesenchymal transition abilities through NLRP3 inhibition in pancreatic cancer [
21]. In this study, we identified the upregulation of DLX6-AS1 in pancreatic cancer. Besides, knockdown of DLX6-AS1 dramatically impaired pancreatic cancer cell proliferation, migration and invasion.
Recently, lncRNAs was found to function as competing endogenous RNAs to regulate the expression level of miRNA, thus playing crucial role in the development of cancer. For better understanding the mechanisms of DLX6-AS1 in pancreatic cancer, we found that miR-181b was of the downstream targets, and miR-181b has been reported as tumor suppressor in various cancers. In glioblastoma, lower level of miR-181b was correlated with more advanced grade patients. MiR-181b can modulate vascular cell adhesion molecule 1 expression and monocyte adhesion in an epidermal growth factor receptor-dependent pathway [
22]. MiR-181b suppressed cell cycle, cell viability and proliferation through down-regulation of FAMLF in burkitt lymphoma cells [
23]. In breast cancer, ectopic expression of miR-181b inhibited the increase of NF-κB level induced by chemokine ligand 18 in cancer cells, thus suppressing cell survival rate and migration [
24]. Consistent with the above reports, our results showed that miR-181b functioned as a tumor suppressor in pancreatic cancer. Its inhibition reversed the reduction of cell viability, migration and invasion abilities caused by DLX6-AS1 knockdown.
Furthermore, we investigated the molecular basis of miR-181b through TargetScan and found that ZEB2 was the downstream target of miR-181b. Here we found that the expression level of ZEB2 in pancreatic cancer tissues was higher than that in the paired adjacent ones. ZEB2 belongs to the ZEB proteins family, and was reported to interact with SMAD protein, involving TGF-β-induced EMT. During EMT, epithelial cells obtain the motile and invasive abilities typical of mesenchymal cells, which is an important process for cancer metastasis and associated with poor prognosis [
25,
26]. In terms of molecular level, this process is indicated by the expression changes of some markers, including loss of epithelial markers and the gain of mesenchymal markers. ZEB2 is reported to be a repressor of E-cadherin and the cadherin switching, like decrease in E-cadherin and increase in N-cadherin is a feature of EMT in malignant tumors [
27,
28]. In the present study, in vitro data showed that DLX6-AS1 knockdown decreased ZEB2 expression level, followed by the increase of E-cadherin and decrease of vimentin and N-cadherin, which was abrogated by miR-181b inhibitors. In the mouse xenograft models, the mRNA level of ZEB2, vimentin and N-cadherin was downregulated while E-cadherin was up-regulated in the DLX6-AS1 knock-down group with reduced tumor size and suppressed tumor metastasis. Taken together, the effect of DLX6-AS1 on EMT depends on the regulation of ZEB2 through targeting miR-181b. In addition, our results showed that the effects of DLX6-AS1 knockdown on survival were marginal, and the effects of DLX6-AS1 knockdown and the rescue experiments have a greater effect on cell migration and EMT-related markers expression, indicating that dysregulation of DLX6-AS1 may be more important for pancreatic cancer metastasis rather than tumor maintenance.
Authors’ contributions
HC designed and conceived the whole study. YA and XC wrote the main manuscript. YA, XC and FM performed the experiments. YY, FM, YJ and DS performed data analysis. All authors contributed to manuscript revisions. All authors read and approved the final manuscript.