Background
Pancreatic cancer (PC) is one of the deadliest tumors with a very low 5-year survival rate (ranges from 2 to 9%) [
1]. Although surgical resection provides a potential cure, about 70% of patients still develop early recurrence within 6–12 months following surgery [
1]. Obviously, the identification of the molecular mechanisms in the initiation and progression of PC is critical for the development of various strategies for PC.
Long non-coding RNAs (lncRNAs) are dysregulated in multiple human cancers including PC [
2], and have been implicated in the control of cellular proliferation, apoptosis, differentiation, migration and invasion. LncRNAs can act as signals, decoys, guides, scaffolds or competing endogenous RNAs (ceRNAs) to modulate gene expression [
3]. DIO3OS is an antisense lncRNA transcribed from the DIO3 gene imprinted locus [
4]. However, the role and the molecular mechanisms of DIO3OS in PC remain to be delineated.
In the present study, we found that lncRNA DIO3OS was significantly upregulated in PC tissues and PC cell lines. Moreover, knockdown of DIO3OS expression suppressed PC cell proliferative and invasion, while overexpression of DIO3OS in PC cells was sufficient to stimulate cell proliferation and invasion. Upon further mechanistic examination, we revealed that DIO3OS served as a molecular sponge for miR-122 to upregulate the expression of ALDOA, which promoted PC cell proliferation and invasion.
Methods
Cell culture and transfection
Human PC cell lines (AsPC-1, MIA PaCa-2, PANC-1 and BxPC-1) and human pancreatic duct epithelial cell line HPDE6-C7 were purchased from the American Type Culture Collection (Rockville, USA). The cells were grown in RPMI1640 or DMEM (Gibco, USA) supplemented with 10% fetal bovine serum (FBS). Plasmids containing DIO3OS and ALDOA, or an empty vector pcDNA3.1 were obtained from Genepharma (Shanghai, China). DIO3OS siRNA, control siRNA miR-122 mimic, control mimic, miR-122 inhibitor and control inhibitor were purchased from IGEbio (Guangzhou, China). Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) was used for cell transfection following the manufacturer’s instructions.
Real-time quantitative PCR (qRT-PCR)
Total RNA was isolated from cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and then was converted to cDNA using an M-MLV Reverse Transcriptase Kit (Invitrogen, Carlsbad, CA, USA). Real-time PCR analysis was carried out using the SYBR-Green-quantitative real-time PCR Master Mix kit (Toyobo, Osaka, Japan). The primers for DIO3OS and GAPDH have been reported [
5]. GAPDH served as the endogenous control. For detecting miRNA expression, the mirVanaTM qRT-PCR microRNA Detection Kit (Ambion Inc., Austin, TX, USA) was used according to the manufacturer’s instructions. MiR-122 expression was normalized to U6.
Western blot analysis
Total protein from cells was extracted using RIPA buffer (Beyotime). An equal amount of each protein sample was separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore, Bedford, MA, USA). The membranes were blocked with 5% nonfat milk at room temperature for 1 h and incubated with specific primary antibody, ALDOA (1:2000, Abcam, Cambridge, UK) and GAPDH (1:5000, Santa Cruz, CA, USA) overnight, followed by incubation with HRP-conjugated secondary antibodies (Santa Cruz, CA, USA). The protein bands were detected using ECL western blotting kit (Amersham Biosciences, Buckinghamshire, UK). GAPDH was used as the loading control.
Cell proliferation assay
Cell proliferation was measured by performing the CCK-8 assay (Beyotime Institute of Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. 5000 cells were seeded into a 96-well plate and were transfected as indicated. Then, cell proliferation was measured 72 h after transfection. The absorbance was measured at 450 nm by a microplate reader (Bio-Rad, Hercules, CA, USA).
A total of 1000 cells were seeded in 24-well plates. After culturing for 14 days, colonies were fixed with 100% methanol for 15 min and then stained with 0.5% crystal violet for 20 min. Colonies with > 100 cells were counted and analyzed.
Cell invasion assay
Transwell invasion assay was performed according to the method described previously [
6,
7]. Briefly, 2 × 10
4 cells in serum-free medium were plated into the upper chamber. The medium containing 10% FBS was added to the lower chamber. After culturing for 24 h, Cell that had invaded were fixed with 75% methanol and stained with crystal violet. Evaluation of invasive capacity was performed by counting invaded cells under a microscope, and five random fields of view were analyzed for each chamber.
Luciferase reporter assay
A luciferase reporter assay was performed as previously described [
8]. The fragment from wild-type DIO3OS (DIO3OS-WT) containing the predicted miR-122-binding site, mutant DIO3OS (DIO3OS-MUT), the 3′-UTR fragment from wild-type ALDOA 3′-UTR (ALDOA-WT) containing the potential miR-122-binding site and mutant ALDOA 3′-UTR (ALDOA-MUT) were amplified using PCR and sub-cloned into a pMIR-GLO Luciferase vector (Promega, Madison, WI, USA). PC cells were co-transfected with the above luciferase reporter vectors containing DIO3OS (WT or MUT) or ALDOA 3′-UTR (WT or MUT) together with miR-122 mimic, miR-122 inhibitor or their negative controls using Lipofectamine 2000 (Invitrogen). The relative luciferase activity was measured with the Dual-Luciferase Reporter Assay System (Promega, China) after 48 h.
RNA immunoprecipitation (RIP) assay
RNA immunoprecipitation assay was conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) as previously described [
9,
10]. Briefly, anti-Argonaute2 (Ago2) antibody (Millipore, Bedford, MA, USA) or normal mouse IgG (Millipore) as a negative control were conjugated to magnetic beads and were incubated with the cell extract in RIP buffer. The immunoprecipitated RNAs were isolated and were subjected to qRT-PCR analysis of DIO3OS and miR-122 expression.
Xenograft assay
All animal procedures were approved by the Institutional Animal Care and Use Committee of First Affiliated Hospital of Zhengzhou University. Female BALB/c nude mice (4 weeks old) were purchased from Beijing HFK Bioscience (Beijing, China) and maintained under pathogen-free conditions.
To evaluate the in vivo tumorigenic effects, MIA PaCa-2 and AsPC-1 cells (2 × 106) were inoculated subcutaneously in the right flank of the nude mice (n = 4 per group). After implantation for 6 days, tumor volume measurement began and was performed every 3 days, using the following formula: volume = length (mm) × width2 (mm2)/2. After 3 weeks, the mice were sacrificed and the tumors were collected for immunohistochemistry assay. The xenografts tissues were formalin-fixed/paraffin-embedded and cut into 4 μm slides. The primary antibody used was anti-Ki-67 (1:1000, Abcam, Cambridge, UK). The secondary streptavidin–horseradish peroxidase-conjugated antibody staining was performed at room temperature, visualized in 3, 3′-diaminobenzidine (ZLI9018, ZSGBBIO, China).
Statistical analysis
Statistical analyses were performed using SPSS 17.0 statistical software (SPSS, Chicago, USA). All data are expressed as the mean ± standard deviation. The Student’s t-test or one-way ANOVA test was used to analyze the significant differences. All experiments were done at least three times. P-value < 0.05 was considered to be statistically significant.
Discussion
Mounting pieces of evidence showed that lncRNAs play important roles in tumor progression and might be used as a diagnostic and therapeutic target. DIO3OS is a newly identified lncRNA [
4]. In the current research, we found for the first time that DIO3OS markedly promoted PC cell proliferation and invasion in vitro and facilitated PC growth in vivo. These data suggested that DIO3OS played a critical role in the biological function of PC development.
LncRNAs might exert their functions as ceRNAs [
3]. Thus, to explore the mechanism by which DIO3OS functions in PC, we searched for candidate miRNAs and confirmed that DIO3OS could compete with miR-122 and other miRNAs to tumor growth and invasion. The potential miRNAs that might interact with DIO3OS should be investigated in future works.
The expression of lncRNAs can be regulated both at the transcriptional and post-transcriptional levels [
14]. In addition, miRNAs can regulate the expression of lncRNAs for degradation by targeting them for degradation via the RNA-induced silencing complex [
15]. However, the detailed mechanisms that can explain DIO3OS induction in PC cells are unclear. In this study, we reported that miR-122 directly bond to DIO3OS and inhibited its expression in PC cells. Therefore, our study supported a DIO3OS-miRNA feedback loop, which enhanced DIO3OS expression and reduced miR-122 expression, leading to the acceleration of PC progression.
Recent studies demonstrated that miR-122 inhibited cell proliferation, migration, and invasion in several tumors [
16‐
19]. Although miR-122 was shown to be downregulated in PC [
20], its role in PC cells remains unclear. Our data support that miR-122 served as a novel tumor suppressor in PC by controlling the expression of DIO3OS. The mechanisms by which miR-122 suppresses PC progression, remains largely elusive.
Abnormal metabolism has been widely regarded as a general hallmark of human cancer, and cancer cells frequently exhibit increased glycolysis and depend largely on this metabolic pathway for the generation of energy, even in the presence of oxygen [
21]. ALDOA, a glycolytic enzyme that catalyzes the reversible conversion of fructose-1, 6-bisphosphate to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate, was shown to be aberrantly expressed in multiple cancer types [
22]. However, accumulated studies have proven that ALDOA also promotes cancer growth and metastasis through its non-enzymatic functions [
22]. In PC cells, ALDOA has been shown to promote the proliferation and metastasis of PC cells [
23]. Our current study confirmed that ALDOA played critical roles in PC cells, in particular, by controlling cell proliferation and invasion. Importantly, we found that ALDOA was a direct target of miR-122. This finding was consistent with the previous notion that miR-122 can inhibit glycolysis and spheroid formation at least by targeting PDK4 in hepatocellular cancer [
24]. It would be interesting to further determine the possible roles of the DIO3OS/miR-122/ALDOA axis in regulating glycolysis and cancer progression in PC.
Conclusions
In conclusion, our study revealed that DIO3OS is upregulated in PC and can promote the proliferation and invasion of PC cells via the miR-122/ALDOA axis. Therefore, the DIO3OS/miR-122/ALDOA axis has potential as an effective therapeutic target against PC.
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