Background
Pancreatic cancer (PC) is a highly invasive malignancy, which is the fifth leading cause of cancer-related deaths with a poor 5-year overall survival rate (<6%) [
1]. Over the past few decades, there has been a marked increase in the incidence of PC [
2]. Due to lack of obvious symptoms at the early stage of PC and a high rate of invasion of blood vessels, lymph nodes, and nerves, treatment through surgical resection is possible in less than 20% of PC cases [
3]. Furthermore, pancreatic fibrosis and other factors lead to resistance to chemotherapy [
4]. For these reasons, identification of novel therapeutic and prognostic biomarkers is a preferred approach for the development of diagnosis and treatment of PC. Several molecules have been recently reported to be crucial for therapy and prognosis of PC, such as MUC-4, LSD1, and FHL2 [
5‐
7].
PEG10 (also known as EDR, HB-1, Mar2, MEF3L, Mart2, and RGAG3) is a paternally expressed imprinted gene which was first reported by Ono R et al. in 2001 [
8]. PEG10 is expressed not only in brain, kidney, and lung tissues in adults but also in embryonic tissues, such as placenta [
9]. Multiple functions have been attributed to this gene, for example, it participates in cell proliferation and differentiation [
10,
11], and inhibition of cell apoptosis [
12]. In addition, some studies have shown that PEG10 was generally overexpressed in several kinds of malignancies, such as lung cancer, hepatic cancer, and B-cell chronic lymphocytic leukemia [
13‐
15]. The overexpression of PEG10 is significantly associated with the proliferation, progression, prognosis, and metastasis of such malignancies.
However, the levels and role of PEG10 in patients with PC have not been investigated extensively. Therefore, in the present study we have assessed the expression of PEG10 in pancreatic cancerous tissues and paired adjacent non-cancerous tissues by immunohistochemistry (IHC). We have also evaluated whether there is a correlation between PEG10 expression and clinicopathological features of PC patients as well as their survival rate. Further, the role of PEG10 in progression of PC cells was also evaluated.
Methods
Patients
PEG10 mRNA expression for 178 pancreatic cancer patients were downloaded from The Cancer Genome Atlas (TCGA) data portal (
http://cancergenome.nih.gov/). A total of 206 formalin-fixed, paraffin-embedded tissues, including 103 cancerous tissues and 103 paired adjacent non-cancerous tissues were obtained from patients diagnosed with PC at Pancreas Center, The First Affiliated Hospital of Nanjing Medical University from 2005 to 2012. All patients received routine preoperative preparation and surgical therapy. The cancerous tissues and paired adjacent non-cancerous tissues were respectively integrated into relevant tissue microarray used for IHC using microarray punching instrument. Our study was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. Written informed consents were obtained from all patients undergoing surgery.
Reagents and antibodies
Anti-human PEG10 antibody was obtained from Novus (Colorado, USA). Anti-human CDK4, CDK2, p21, p27, SKP2, and E2F-1 antibodies were purchased from Cell Signaling Technology (CST, Massachusetts, USA). Anti-human Cyclin E1 antibody was obtained from Abcam. Si-RNAs for PEG10 and E2F-1 and respective negative controls were purchased from GenePharma. Annexin V PE apoptosis detection kit was purchased from eBioscience (Hatfield, UK) and PI/RNase staining buffer was obtained from BD biosciences (New York, USA). Cell counting kit-8 (CCK-8) and EDU kit were obtained from Dojindo and RIBOBIO, respectively. Trizol reagent and PrimeScript RT Master Mix (Perfect Real Time) were both obtained from TaKaRa (Shiga, Japan) and FastStart Universal SYBR Green Master was purchased from Roche.
Cell lines and cell culture
Pancreatic cancer cell lines AsPC-1, Mia PaCa-2, SW1990, BxPc-3, Capan-2, CFPAC-1, PANC-1 were available in our laboratory. Normal human pancreatic ductal cell line hTERT-HPNE was purchased from the American Type Culture Collection (ATCC; Rockville, MD, USA). Pancreatic cancer cell lines were cultured in DMEM supplemented with 10% FBS, penicillin (100 U/mL) and streptomycin (100 μg/mL). hTERT-HPNE cell line was cultured according to the recommendation of ATCC.
IHC analysis
Immunohistochemical staining was performed using a standard immunoperoxidase staining procedure. The tissue sections were viewed independently by two experienced pathologists, who were blinded to the clinicopathological information and clinical outcomes of the patients enrolled in our study. The percentage of PEG10 positive cells was scored as follows: 0 (0%), 1 (1%), 2 (2%) …… 99 (99%), 100 (100%). The staining intensity was visually scored as follows: 0 (negatively stained), 1 (weakly stained), 2 (moderately stained), and 3 (strongly stained). Both the percentage of positive cancer cells and staining intensity were decided independently by double-blinded manner. IHC score for each case was calculated by the following formula: IHC score = positive rate score × intensity score. All patients were divided into three groups (high, medium, and low) according to the tri-sectional quantiles of PEG10 IHC score. And then, cases with IHC score >50 (high and medium) were regarded as high expression of PEG10; on the contrary, cases with IHC score ≤50 (low) were regarded as low expression.
Small interfering RNA and plasmid vector related assays
Cells were seeded in 12-well plates (1.8 × 105 cells/well) and cultured in complete medium for 12 h. After replacing fresh complete medium, Si-RNAs (or plasmid vector) and negative controls were added into relevant wells using jetPRIME® transfection (Polyplus, NY, USA) according to the manufacturer’s instructions. Following transfection for 48–72 h, cells were collected for subsequent experiments.
Quantitative real time reverse transcription polymerase chain reaction
Total RNA was extracted from different cell lines using Trizol reagent and reverse-transcribed into cDNA with PrimeScript RT Master Mix. The process of qRT-PCR amplification was performed using the Step One Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) with FastStart Universal SYBR Green Master. All the experiments mentioned here were performed according to relevant manufacturer’s instructions. The specific primers for human PEG10, E2F-1, and β-ACTIN were designed by Primer Premier 5 and checked by Oligo 6. The formula 2-ΔΔCt (Ct means the cycle threshold) was used to normalize the relative expression of PEG10 mRNA in certain cells.
Western blotting
Total protein was extracted by using a lysis buffer containing PMSF, protease inhibitors, and phosphatase inhibitors (1 mL lysis buffer with 5 μl 100 mM PMSF, 1 μL protease inhibitors, and 5 μL phosphatase inhibitors). Protein lysates from cells were subjected to 5× SDS-PAGE. Western blot analysis was performed by using standard methods.
Proliferation assays
CCK-8 assay
Cells transfected with siRNA for PEG10 and negative controls were plated in 96-well plates at a density of 2.5 × 103 cells/well. After culturing for 24 h, 100 μL complete medium containing 10 μL CCK-8 reagent was added to each well. Similarly, 100 μL completed medium containing 10 μL CCK-8 reagent was added to respective wells at different time points (24 h, 48 h, 72 h, 96 h, and 120 h). The plates were incubated in dark at 37 °C for 2 h and analyzed at 450 nm absorbance. At least three wells were assessed for each group.
Different cell lines were seeded in 6-well plates (6 × 102 cells/well). The culture medium was changed every 2 days. The cells were stained with crystal violet after culturing for 10 days. The number of clones was then counted to evaluate cell proliferation.
EDU assay
Cells that had undergone different interventions were seeded in 96-well plates at a density of 3 × 103 cells/well. EDU assay was done as per manufacturer’s instructions following 48 h culture. Cell proliferation was assessed by calculating percentage of positive stained cells.
Apoptosis detection
Cells in each group were harvested after 48 h culture and resuspended in 500 μL Annexin V binding buffer. The cells were stained with 5 μL PE Annexin V and 7-AAD Viability Staining Solution and incubated in dark at room temperature for 15 min before being analyzed by flowcytometer (Gallios, Beckman Coulter, USA).
Cell cycle detection
Cells transfected with Si-RNA for PEG10 and negative controls were cultured in serum-free medium for 24 h and complete medium for 48 h. Harvested cells were fixed in 70% formaldehyde at 4 °C for 5 h and stained with 500 μL PI/RNase staining buffer. Stained cells were then analyzed by flowcytometer.
Xenograft tumorigenicity assays
All female BALB/c nude mice aged 5 weeks used in this study were purchased from Model Animal Research Center of Nanjing University. To assess the function of PEG10 in vivo, fresh cancer cells (1 × 106 per mice) were implanted into the subcutaneous tissues which were induced by 1% pentobarbital sodium. The mice were randomly divided into two groups after four weeks of tumor implantation. For each group, intratumoral injection with Si-RNA or negative control was performed once every 48 h. Si-RNA and reagent (in vivo-jetPEI, Polyplus, NY, USA) were separately added in 5% glucose solution, and mixed them together. After a 15 min incubation time at room temperature, the Si-RNA/ in vivo-jetPEI® complexes were injected into animal. After 4 weeks, the mice executed humanely and the tumors were excised and photographed.
Migration and invasion assays
Cell migration and invasion was assessed using transwell filters purchased from BD Biosciences (Franklin Lakes, NJ). PANC-1 (3 × 104) and CFPAC-1 (3 × 104) cells cultured in serum-free medium for 24 h were seeded into the upper chamber containing an uncoated or Matrigel-coated membrane and 200 μL serum-free medium. Complete medium (500 μL) was added to the lower chamber. Cells that migrated to the lower compartment were stained with crystal violet after 24 h of incubation at 37 °C in a humidified 5% CO2 incubator. Three fields in each well were randomly chosen to count migrated and invaded cells.
Chromatin immunoprecipitation (ChIP)
ChIP assay was carried out using an EZ-Magna ChIP kit (Millipore, Darmstadt, Germany) according to the manufacturer’s protocol. Briefly, 2 × 106 cells were treated with 1% formaldehyde for 10 min for crosslinking, and then quenched by adding 0.125 M glycine. The cells were scraped with PBS and collected after 5-min centrifugation at 800 × g at 4 °C. Then, the cross-linked cells were resuspended in 1% SDS lysis buffer, and the soluble chromatin was sheared into 400-bp fragment DNA using an Ultrasonic Cell Disruptor (Covaris, USA). The fragmented chromatin samples were aliquoted as genomic input DNA or immunoprecipitated with 5 ug E2F-1 antibodies, or rabbit IgG, and incubated at 4 °C with rotation for 16 h. Immunocomplexes collected by magnetic separator were washed and eluted with 1% SDS and 0.1 M NaHCO3, and DNA was purified on spin columns. The ChIP products and genomic input DNA were quantitatively analyzed by real-time PCR (E2F-1 primer sequences: forward, 5′- CCTGGAATTATTCTATCTTGCAGAA-3′; reverse, 5′- AATGAATGAAATGCAGCTTTTTAAC-3′). ChIP data are presented as the percentage of input normalized to control purifications.
Statistical analysis
The paired Student’s t-test was applied to compare PEG10 expression in pancreatic cancerous and paired non-cancerous tissues. The association of PEG10 expression with clinicopathologic features was analyzed by the Pearson χ2 test. Survival analysis was assessed by Kaplan Meier plots and log-rank tests. Independent prognostic factors were recognized through univariate and multivariate Cox proportional hazard regression models. The comparison between two groups was done by independent Student’s t-test. Stata 10.0 software was applied to process survival related analysis, and SPSS 20.0 software were used to perform others. All data were expressed as mean ± SD. Differences were considered statistically significant at P < 0.05.
Discussion
In this study, we show that PEG10 was abnormally overexpressed in PC and was significantly associated with PC progression and prognosis. Furthermore, E2F-1 mediated PEG10 upregulation could promote PC cell proliferation, migration, and invasion. Thus, we identified PEG10 as a potential prognostic and therapeutic target for PC.
PEG10 is a paternally expressed imprinted gene, which was reported to have been derived from the Ty3/Gypsy family of retrotransposons [
20]. Similar to many other imprinted genes, the function of PEG10 is decided by its expression levels or methylation level [
21‐
23]. In our study, we focused on the altered expression of PEG10 in PC. During physiological conditions, adequate PEG10 expression is necessary for placental development and it has been shown that PEG10 is downregulated in case of preeclampsia [
24‐
26]. PEG10 expression was later shown to be abnormally reactivated in some malignancies (such as liver cancer, lung cancer, and gallbladder cancer) and that it could be a biomarker for progression and prognosis of certain cancers [
13,
27,
28]. The findings of our study show for the first time that PEG10 is highly expressed in PC and is associated with tumor size, vessel invasion, and shorter overall survival time. Cox proportional hazard regression analysis further illustrates that PEG10 is an independent factor for poor prognosis of PC.
Furthermore, we also propose the role of PEG10 in PC cell proliferation and the underlying mechanism. PEG10 has been reported to promote cancer cell proliferation through mechanisms described as follows. PEG10 overexpression was reported to be associated with a mediator of apoptosis (SIAH1) resulting in decrease in cell death as in hepatocellular carcinomas [
12]. In neuroendocrine prostate cancer, PEG10 knockdown was shown to suppress cell cycle progression through upregulation of p21 and p27 and downregulation of CDK2 [
19]. In the present study, we also investigated the proliferation promoting effect of PEG10 considering these two mechanisms; and the results were similar to the previously published studies. Cell apoptosis rate in PEG10 knockdown PC cells was similar to that in control cells. This phenomenon may due to the inefficiency of PEG10 mediated anti-apoptosis effect or the presence of other apoptosis regulatory mechanisms. However, the G0/G1 phase arrest in PEG10 knockdown PC cells was obviously observed. Further molecular mechanism analysis suggested that expression of p21 and p27, which are known cyclin-dependent kinase inhibitors, were significantly increased in cells with PEG10 downregulation. These molecules could inhibit the activation of CDK2 and CDK4 through binding to them, as reported previously [
29,
30]. However, the expression of CDK4 was not significantly affected in cells with PEG10 downregulation and control cells. Furthermore, the expression of SKP2 was negatively associated with p27 which was similar to the results of previous studies in pancreatic cancer [
31,
32]. Data related to cell cycle were consistent with the studies described above.
Though the roles of PEG10 in PC cell proliferation, apoptosis, and cell cycle have been investigated in the present study, the roles of this protein in other aspects are relatively unknown. In previous studies, it has been reported that PEG10 could induce migration of Burkitt’s lymphoma cells via upregulation of MMP-2 and MMP-9 [
33]. PEG10 has been reported to promote lung cancer cell migration and invasion by upregulating the expression of β-catenin, MMP-2 and MMP-9, and decreasing the expression of E-cadherin [
13]. Further, PEG10 was shown to trigger prostate cancer cell invasion by enhancing Snail expression via TGF-β signaling [
19]. Our findings show that PEG10 expression is positively associated with vessel invasion in PC, and PEG10 promotes PC cells migration and invasion through ERK/MMP7 pathway. The MMP2 mRNA was both decreased in two pancreatic cancer cell lines after PEG10 knockdown. However, the protein of MMP2 was only decreased in one of the cell lines (CFPAC-1). Therefore, these data was not sufficient to suggest that MMP2 was regulated by PEG10.
Although downstream events of PEG10 in PC have been discussed preliminarily, the upstream events of this protein are still unclear. E2F-1 is a member of the E2F family of transcription factors, which plays a crucial role in the control of cell cycle. E2F-1 has been reported to regulate PEG10 expression via directly binding to the promoter of PEG10 in liver and prostate cancer [
18,
19]. Similar to these studies, our ChIP and western blotting assay results also demonstrated that E2F-1 could interact with PEG10 promoter to further upregulate PEG10 expression. Furthermore, PEG10 could also be regulated by some other factors, such as c-MYC, CXCL13, CCL19, and androgen receptor. Li et al. revealed that c-MYC, which serves as a classic proto-oncogene, could act directly upstream of proliferation-positive gene PEG10 [
10]. Levels of PEG10 expression in freshly isolated B-ALL and B-CLL CD19
+CD34
+ B cells has been shown to be significantly increased after stimulation with CXCL13 and CCL19 together [
34]. The upregulation of PEG10 in this pattern may be involved in the resistance to TNF-α-mediated apoptosis of B cells. Moreover, the interaction between androgen and androgen receptor was firstly described as a facilitating factor for PEG10 expression in hepatic cancer [
35], and then confirmed in gastric cancer [
36]. Whether the factors discussed above participate in the activation of PEG10 in PC needs to be further investigated.
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (81170336, 81272239, 81672471). This work was also supported by Talents planning of six summit fields of Jiangsu Province (WSW-032), the Innovation Capability Development Project of Jiangsu Province (BM2015004), the Research Innovation Program for College Graduates of Jiangsu Province (KYLX15_0954) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD, JX10231801).
We thank Elixigen Corporation (Huntington Beach, California, USA) for helping in proofreading and editing the English of final manuscript.