Background
Pancreatic cancer is one of the poorest prognoses among human tumors, with an overall 5-year survival rate of less than 5% [
1]. In spite of the continuous developments in clinical therapies and novel surgical techniques, the survival of patients with pancreatic cancer has remained poor for over 30 years [
2]. Pancreatic cancer is highly aggressive, wherein tissue invasion and remote metastasis may occur during the early stages [
3]. Because invasion and metastasis are the most formidable obstacles preventing the effective treatment of pancreatic cancer, it is therefore essential to explore the molecular mechanisms that contribute to this aggressive behavior, in order to improve the outcomes for patients.
miR-663, a member of the primate-specific miRNA family, is associated with many important biologic processes, including the evolution, development, viral infection, inflammatory responses, and carcinogenesis of many vertebrate species [
4-
9]. However, its role in tumor progression can be considered quite contradictory. Although it acts as an oncogene that promotes the malignancy of lung cancer, nasopharyngeal carcinoma, and breast cancer [
6,
7], miR-663 may also act as a potential tumor attenuate molecular in gastric cancer, colorectal carcinoma, prostate cancer, and acute lymphoblastic leukemia [
8,
9]. Overall speaking, the effect and mechanism of miR-663 on pancreatic cancer remain unclear.
Eukaryotic elongation factor 1-a (eEF1A) is a member of the G protein family, and one of the four subunits that constitute the eukaryotic elongation factor 1 [
10,
11]. And it has two identified isoforms, namely eEF1A1 and eEF1A2. Unlike eEF1A1, which is expressed almost ubiquitously, eEF1A2 is normally only present in the heart, brain, and skeletal muscles [
12-
14]. eEF1A2 is involved in a variety of biological processes such as cytoskeleton modification [
13,
14], targeting proteins for degradation [
15], heat shock response [
16], apoptosis [
17], and phosphatidylinositol signaling [
18,
19]. eEF1A2 is considered as an oncogene marker because it is highly expressed in a subset of cancers, such as ovarian cancer [
20], breast cancer [
21], and pancreatic cancer [
22].
In the present study, we evaluated the expression and clinical relevance of miR-663 and eEF1A2 in pancreatic cancer. Additionally, we investigated the functional role of miR-663 in the invasion of pancreatic cancer cell lines. The contribution of miR-663 to pancreatic cancer malignancy and the underlying molecular mechanisms were also investigated. Our data demonstrate that miR-663 has potential value as a prognostic marker and as a therapeutic target of pancreatic cancer.
Discussion
miR-663 is expressed in Homo sapiens and Pan troglodytes. miR-663 belongs to the primate-specific miRNAs that possibly attribute to the vertebrates’ evolution, development, and carcinogenesis [
31-
34]. The effect of miR-663 in malignant progression is controversial, because it could act as a tumor attenuate molecular or promoter in an organ-specific fashion [
6,
7]. The clinical relevance of miR-663 in pancreatic cancer remains unknown. Our present study represents the first comprehensive analysis of miR-663 in pancreatic cancer. We selected miR-663 as the target molecule from miRNA profiling, and then identified miR-663 as a tumor attenuate molecular that can attenuate the proliferation and invasion of pancreatic cancer. Mechanistically, we identified eEF1A2 as a direct and functional target of miR-663, which deepened our understanding of the mechanisms underlying pancreatic cancer progression.
Our study has demonstrated that eEF1A2 is significantly upregulated and miR-663 is significantly downregulated in pancreatic cancer. The additional results showed that eEF1A2 and miR-663 expression level were significantly associated with TNM stage and node metastasis status of the patients, but not associated with the patient’s age or gender, or with the tumor’s diameter, location or differentiation. Furthermore, while the tumor’s diameter, differentiation, clinical stage and lymph node metastasis were significantly associated with the survival of patients, the gender, age, and location were not. Since eEF1A2 and miR-663 are both associated with the survival of patient in pancreatic cancer, both may be potential biomarkers for prognosis in patients with pancreatic cancer.
Most importantly, we found that miR-663 and eEF1A2 were not only correlated inversely with each other, but were also predictive of the survival of pancreatic cancer patients. These findings highlight the potential values of miR-663 and eEF1A2 as novel prognostic biomarkers in human pancreatic cancer.
Pancreatic cancer is characterized by rapid growth and relentless invasion. Invasiveness and metastasis, the two leading causes of cancer mortalities, are especially pronounced in pancreatic cancer, which shows strikingly high invasive and metastatic potential [
3]. We highlighted the therapeutic potential of miR-663 in pancreatic cancer treatment with our identification of miR-663 as a tumor attenuate molecular that attenuates the proliferation and invasion of pancreatic cancer cells both in vitro and in vivo, and that attenuates of growth and invasion of pancreatic cancer. MMPs regulate basic cellular processes including survival, migration and morphogenesis and degradation extracellular matrix during the cancer metastatic process [
23]. Akt plays a key role in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, transcription and cell migration [
27]. We examined the expression levels of MMP9, Akt and pAkt(Ser473), and then found that the ectopic expression of miR-663 markedly attenuated the protein expression of MMP9, Akt and pAkt, in PANC-1 and AsPC-1 cells concomitant with the attenuation of cell invasion. These results suggest that miR-663 attenuates pancreatic cancer cell invasiveness, a property likely associated with MMP9 and Akt.
Eukaryotic elongation factor 1 alpha (eEF1A) is critical for protein synthesis because it regulates ribosomal polypeptide elongation by binding to amino-acylated tRNAs and by facilitating recruitment to the ribosome [
35]. The two isoforms of eEF1A identified in humans, namely, eEF1A1 and eEF1A2, are encoded by distinct genes. eEF1A2 is a protein translation factor with important functions in tumor genesis and progression, for it is frequently overexpressed in various cancers. One recent study had demonstrated that eEF1A2 promoted cell invasion in pancreatic cancer by upregulating the expression of MMP-9 through Akt activation [
22]. We identified eEF1A2 as a direct target of miR-663 by bioinformatics prediction, signal network analysis and Dual-luciferase assay. The 3′ untranslated region (3′UTR) of eEF1A2 contains a seed region for miR-663. Our pancreatic cancer tissue study has showed that eEF1A2 was significantly upregulated and miR-663 was significantly downregulated in pancreatic cancer. Most importantly, we found that miR-663 and eEF1A2 were inversely correlated with each other. These results further supported the conclusion that miR-663 targets eEF1A2.
Additionally, further study indicated that the expression of eEF1A2 could partially restore the pro-apoptotic and anti-invasion functions of miR-663. The knockdown of eEF1A2 and the overexpression of miR-663 produce similar biological effects on pancreatic cancer cells, with the effect being more obvious in PANC-1 cells overexpressing miR-663. The results revealed that the attenuation effect of miR-663 on pancreatic cancer was due at least in part to its targeting of eEF1A2, at the same time there were other targets taking part in the process. Our study has demonstrated the potential of miR-663 as a target for future pancreatic cancer treatments.
In summary, the study revealed that eEF1A2 was significantly upregulated and miR-663 was significantly downregulated in pancreatic cancer, and that both EEF1A2 and miR-663 are associated with the survival of pancreatic cancer patients. Moreover, miR-663 and eEF1A2 were correlated inversely with each other. Specifically, miR-663 attenuated the proliferation and invasion of pancreatic cells in vitro and in vivo by directly targeting eEF1A2. Most importantly, miR-663 and eEF1A2 may prove to be novel prognostic biomarkers and potential targets for future treatments of pancreatic cancer.
Materials and methods
Patients and tissue specimens
The tissue specimens were collected from 68 patients with pancreatic cancer who had undergone either pancreaticoduodenectomy or distal pancreatectomy between 2007 and 2009 at the First Affiliated Hospital of Zhengzhou University and the Henan Tumor Hospital of Zhengzhou University in Zhengzhou, China.
After the frozen specimens were HE stained and examined for clinicopathological features by surgical pathologists, they were then snap frozen in liquid nitrogen. Normal pancreas tissues adjacent to tumors were used as controls. None of the cancer patients in this study had received preoperative radiation or chemotherapy. Informed consent was obtained from the patients. The clinicopathologic features of these patients have been summarized in Table
1. The 68 patients with complete information were followed up after operation until February 1st, 2014, with a median follow-up time of 17.8 months. The study was approved by the Research Ethics Committee of Zhengzhou University.
Cell culture
Human pancreatic cell line HPDE6-C7 and pancreatic cancer cell lines PANC-1, Capan-2, SW1990, BxPC3 and AsPC-1 were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Subsequently, these cell lines were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Australia), 100 U/ml penicillin, and 50 μg/ml streptomycin in incubators with humidified atmosphere of 5% CO2 and 95% air at 37°C.
miRNA transfection
The miR-663 agomir (GMR-miR™ microRNA-663 agomir) used in this study was synthesized by Shanghai GenePharma Co. Ltd. Prior to transfection, cells were plated at a density of 1.5 × 105 cells/well in 6-well plates. Once cells reached ~70% confluence, transient transfection was conducted using Lipofectamine™2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfection efficiencies were evaluated in every experiment by qRT-PCR at 24 h post-transfection. Cells from each cell line were subdivided into three groups: the non-transfected blank group (Blank), the scrambled miR-663 transfected negative control group (NC) and the miR-663 agomir transfected group (miR-663).
Quantitative real-time PCR
Total RNA wasextracted frompancreatic cancer tissue samples and adjacent non-tumor tissue samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. 1 μg RNA was used to synthesize cDNA. And the expression levels of eEF1A2 and miR-663 were determined by qPCR (ABI 7500 fast system, Applied Biosystems, CA, USA). We used U6 small nuclear RNA (U6 snRNA) as an endogenous control for normalization. The qRT-PCR results were expressed relative to miR-663 expression levels at the threshold cycle (Ct) and were converted to fold changes (2-ΔΔCt).
Western blot analysis
Total proteins of cells were extracted using RIPA buffer containing phenylme thanesulfonylfluoride (PMSF). The protein obtained was measured using a BCA Protein Assay Kit (Pierce, Rockford, IL). 30 μg of protein lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto PVDF membranes. PVDF membranes were blocked in 5% BSA in 0.05% tween 20-TBS for 1 h and incubated with primary antibody overnight at 4 diluted in foregoing blocking buffer. Dilutions for primary antibodies were as follows: anti-eEF1A2 (1:1000, Santa Cruz, USA), anti-MMP-9 (1:400, Santa Cruz, USA), anti-total Akt(1:1000, Santa Cruz, USA), anti-p-Akt (Ser 473) (1:1000, Santa Cruz, USA), anti-BAD(1:1000, Santa Cruz, USA) anti-BAX (1:1000, Santa Cruz, USA). After extensive washing with TBST, anti-rabbit IgG-HRP secondary antibody (1:5000, Santa Cruz, USA) was added. Signals were determined using a chemiluminescence detection kit (Amersham Pharmacia Biotech, Piscataway, NJ). An antibody against β-actin (Santa Cruz, USA) served as an endogenous reference.
Cell proliferation assay
The pancreatic cancer cells were transferred to 96-wells plates at a density of 1 × 104 cells/well, with five replicate wells per group. Then the relative numbers of viable cells were detected by Cell Counting Kit-8 reagents (CCK-8; Dojindo, Japan) after 0, 1, 2, 3 days of cultivation. The results were recorded using a microplate reader (Elx800; BioTek, VT, USA), in absorbance optical density at 450 nm. The experiments were independently triplicated.
Human pancreatic cancer cell linesPANC-1 and AsPC-1 were seeded and transfected with miR-663. Cells were suspended in RPMI-1640 containing 0.35% low melting agarose, and plated onto solidified 0.6% agarose containing RPMI-1640 in six-well culture plates at a density of 1 × 105 cells per dish. The plates were incubated for 2 weeks at 37 in a 5% CO2 incubator, and the number of colonies was counted after staining with 0.1% crystal violet solution. The colonies of more than 50 cells were manually counted. The experiments were independently triplicated.
Transwell assay
Transwell filters (Costar, USA) were coated with matrigel (3.9 μg/μl, 60–80 μl) on the upper surface of the polycarbonic membrane (6.5 mm in diameter, 8 μm pore size). After 30 min of incubation at 37°C, the matrigel solidified and served as the extracellular matrix for tumor cell invasion analysis. Cells of the transfected and control groups (1 × 105) were harvested in 100 μl of serum free RPMI-1640 medium and added to the upper compartment of the chamber. After 24 h of incubation at 37°C and 5% CO2, the medium was removed from the upper chamber, and then stained with methylene blue Staining Solution (Beyotime). Noninvasive cells on the upper surface were wiped with a cotton swab. The number of cells invading the matrigel was counted from three randomly selected visual fields, each from the central and peripheral portion of the filter, using an inverted microscope at 200× magnification. All experiments were performed in triplicate.
Flow cytometry assay
Cell apoptosis was performed by flow cytometry. Human pancreatic cancer cell lines PANC-1 and AsPC-1 cells were harvested at 48 h post-transfection by trypsinization. Tumor cells were resuspended at a density of 1 × 106 cells/mL in 1 × binding buffer. After double staining with FITC-Annexin V and propidium iodide (PI) using the FITC Annexin V Apoptosis Detection Kit I (BestBio, Shanghai, China), cells were analyzed using a FACScan® flow cytometer equipped with Cell Quest software (BD Biosciences, San Jose, CA, USA) according to manufacturer’s instructions to detect early and late apoptosis of cells. All experiments were performed in triplicate.
Hoechst 33342 staining
Cells were treated with different concentrations of a1-PDX for 48 h and washed twice with cold PBS, and then incubated in the dark in 5 μL Hoechst /PI staining buffer for 15 min at 25°C. The images were recorded on a computer with a digital camera attached to the microscope, and then images were processed by the computer. The Hoechst reagent was taken up by the nuclei of the cells, and apoptotic cells exhibited a bright blue fluorescence. For quantification of Hoechst 33342 staining, the percentage of Hoechst-positive nuclei per optical field (at least 50 fields) was counted. All experiments were performed in triplicate.
Tumor xenograft model of nude mouse
Six-week-old male nude BALB/c mice were purchased from Henan Experimental Animals Centre (Zhengzhou, China). Tumor xenograft model of nude mouse is a reliable in vivo model for tumor study. To assess the effect of miR-663 on tumorigenicity in vivo, we purchased lentivirus expressing pre-miR-663 from Shanghai GenePharma Co. Ltd and infected it into the PANC-1 cells. The 2 × 106 stably transfected cells are subcutaneously injected in the dorsal scapular region of 6-week-old BALB/c nude mice (6 mice/group), which had been anesthetized by inhaling sevoflurane. The tumors formed were measured with a caliper every 7 days, and tumor volume was calculated using the following formula: volume = Л (length × width2)/6. Tumors were harvested after 4 weeks. These protocols were approved by the Zhengzhou University Animal Care and Use Committee.
Dual-luciferase assay
Human eEF1A2 3′ UTR (bases108-115) fragment containing putative binding sites for miR-663 were amplified by PCR from human genomic DNA. The mutant eEF1A2 3′ UTRs was obtained by overlap extension PCR. The fragments were cloned into a pmir-GLO reporter vector (Promega), downstream of the luciferase gene, to generate the recombinant vectors pmir-GLO-eEF1A2-wt and pmir-GLO-eEF1A2-mut. For the luciferase reporter assay, PANC-1 and AsPC-1 were transiently co-transfected with miRNA (miR-633 agomir or miR-633 scramble) and reporter vectors (wild-type reporter vectors or mutant-type reporter vectors), using Lipofectamine™ 2000. Luciferase activities were measured using a Dual-Luciferase assay kit (Promega) according to manufacturer’s instructions at 48 h post-transfection.
Statistical analyses
All statistical analyses were performed using SPSS 17.0 software. The Student t test or one-way ANOVA was conducted for normally distributed data. The Pearson χ
2 test was used to determine the correlation between miR-663 expression and clinicopathologic features of patients. Patients’ survival was analyzed in the Kaplan-Meier method, using the log-rank test for comparison. All data represent mean ± SD. Statistical significance was set at p < 0.05.
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Competing interests
The authors have declared that no competing interest exists.
Authors’ contributions
WQZ and GQZ designed and guided the study. WQZ, YYW, T W, M Land YWD performed and participated in analysis of laboratory experiments data. WQZ and XNC acquired, preserved clinical samples. GQZ provided administrative support and funded experiments. WQZ, YYW and GQZ drafted the manuscript. All authors have contributed and approved the final manuscript.