Background
Pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of all pancreatic carcinomas [
1] and has a poor five-year survival rate of 2–9% [
2] owing to a lack of early symptoms and early detection and most cases being diagnosed at an advanced stage. PDAC tumorigenesis and progression are the result of a series of gene mutations and previous studies have revealed that KRAS, P16, CDNK27, P53, and SMAD4 mutations contribute toward PDAC development [
3,
4]; however, the detailed molecular pathogenesis of PDAC remains largely unknown.
Cell cycle deregulation is a common feature of human cancer [
5], with fundamental mutations in the genetic control of cell division resulting in spontaneous proliferation [
6]. For instance, P16 and P53 mutations release the inhibition of CDK4/CDK6-mediated RB phosphorylation and trigger E2F activation-mediated cell cycle progression [
7,
8]. Aberrant high E2F expression has been reported in many malignant cancers, including gastric, colorectal, breast, and lung cancers, and E2F dysregulation has been shown to contribute toward unrestrained cell proliferation and cancer development [
9].
Long non coding RNAs (lncRNAs), which function primarily via interaction partners such as chromatin DNA, proteins, and RNAs [
10,
11] have recently attracted widespread attention as a new coregulator of cancer development [
12]. Numerous oncogenic and tumor-suppressive lncRNAs have been reported to regulate tumor cell proliferation, growth, metabolism, and metastasis, thus are regarded as potential biomarkers and therapeutic targets for cancer diagnosis and treatment [
13,
14]. Although some E2F1-responsive lncRNAs have been shown to play key roles in cell proliferation and cell cycle transition [
15‐
18], none have directly regulated E2F1 transactivation.
The lncRNA Linc00337 (accession number: NR_103534.1) has been shown to promote gastric cancer, lung cancer, colorectal cancer and esophageal squamous cell carcinoma progression cell proliferation [
19‐
22]; however, it has been poorly studied and its roles and underlying mechanisms in PDAC remain largely unclear. In this study, we conducted a comprehensive survey of cancer related lncRNAs in The Cancer Genome Atlas (TCGA) PDAC database and found that Linc00337 is significantly upregulated in PDAC and predicts poor prognosis. Therefore, we investigated the interaction between Linc00337 and E2F1 and the role of Linc00337 in PDAC by detecting Linc00337 expression in paired PDAC tissues and PDAC cells. Thus, our study suggests that Linc00337 could be used as a potential prognostic biomarker and therapeutic target for PDAC.
Materials and methods
Clinical specimens
Fresh paired PDAC tissues were collected from 18 cases of PDAC surgical resection at the Department of General Surgery, Xinhua Hospital, with informed consent. Normal tissues were defined as that 2 cm away from malignant tissues. The patient inclusion criteria were as follows: (1) aged 46–70; (2) no other types of tumor or diseases; (3) had not undergone preoperative chemotherapy or radiation therapy. All tissues were obtained under sterile conditions during surgery, flash frozen in liquid nitrogen, and stored at − 80 °C.
Cell lines
The normal pancreatic ductal cell line HPDE and PDAC cell lines AsPC1, BxPC3, MiaPaCa2, and PANC1 were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were maintained in DMEM with high glucose and sodium pyruvate, except for AsPC1 which was maintained in 1640. All media were supplemented with 10% fetal bovine serum (Sigma), 100 units/mL of penicillin and 100 μg/mL of streptomycin (Gibco).
Lentiviral infection and establishment of stable cell lines
Linc00337 (pLVX-337, pLVX-E2F1) or empty (pLVX) vectors were purchased from GeneCopoeia (Guangzhou, China), and Linc00337-targeting (sh#1, sh#2, sh#3, shE2F1) or negative control (NC) shRNAs were obtained from RiboBio (Guangzhoug, China). The lentivirus was constructed in HEK-293 T cells and collected from the supernatant after 24 and 48 h. AsPC1 or PANC1 cells were infected with the lentiviruses and selected with 2 μg/mL of puromycin (Millipore, USA) 48 h after infection.
RNA isolation, RNA-seq, quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from tissues and cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer’s instructions and subjected to RNA-seq analysis by Shanghai Kangcheng Biotech. Raw data were deposited in the GEO database (accession number GSE146671). For qRT-PCR, 2 μg of total RNA was treated with DNase I and reverse transcribed using an MMLV system (Promega, USA). qRT-PCR was performed using an ABI 7900 RT-PCR system with SYBR Green Real-time PCR Master Mix (ABI, USA) and 18S RNA as a control. Primer sequences are listed in Table
1.
Table 1
primers and shRNA target sequences
# qRT-PCR primers (5′-3′) |
| Forward | Reverse |
337 V1 | CTCTGATCTGTCCACCCTCG | TTCCTGGGGTTTGTGTTCGG |
337 V2 | GCGAATCATTTGAGTAGAGA | CAAGTGAAGTCAGGATCACA |
337 V1 + V3 | AGTTGCTGGAGTTGCCGAAT | TCTCCGTGTGTGTGTTTCCC |
18S | GTAACCCGTTGAACCCCATT | CCATCCAATCGGTAGTAGCG |
GAPDH | GGAGCGAGATCCCTCCAAAAT | GGCTGTTGTCATACTTCTCATGG |
JUNB | ACAAACTCCTGAAACCGAGCC | CGAGCCCTGACCAGAAAAGTA |
JUND | TCATCATCCAGTCCAACGGG | TTCTGCTTGTGTAAATCCTCCAG |
CCNB1 | AATAAGGCGAAGATCAACATGGC | TTTGTTACCAATGTCCCCAAGAG |
CCNB2 | CCGACGGTGTCCAGTGATTT | TGTTGTTTTGGTGGGTTGAACT |
MYC | GGCTCCTGGCAAAAGGTCA | CTGCGTAGTTGTGCTGATGT |
ChIRP used primers (5′-3′) |
CCNB1 | TGTGACCCTGGCAAAGTCAT | CAAGAGTGTGCGTTGCCAAT |
JUNB | ATGCGTACCCCGAGGTCCTTTGA | AGCCTGAGCCACACGCCTTTATA |
MYC | TTTATAATGCGAGGGTCTGG | TATTCGCTCCGGATCTCCCTT |
FOXM1 | GAGCTTTGAAAAGGGGAGCA | ACCGGAGCTTTCAGTTTGTT |
shRNA target sequenses (5′-3′) |
337 sh#1 | CCTCCCAAAGTGCTGAGATTA | |
337 sh#2 | CCTCCCAAAGTGCTGAGATTA | |
337 sh#3 | GTACCACTTTATTTTATTTTA | |
E2F1 shRNA | AGCTGGACCACCTGATGAATA | |
ChIP-qRT-PCR primers | |
Forward | Reverse |
337BS1 | CCAAGTAGCTAGAATTACAGC | TTTAATCCCAGCACTTTGGGA |
337BS2 | TGCGCACTGTGCCGCCGATGC | CTCCTGAGTTTGGGACTAAGT |
U1 | ATACTTACCTGGCAGGGGA | AGGGGAAAGCGCGAACGCAG |
MYC | AGATCCTCTCTCGCTAATCT | ATACTCAGCGCGATCCCTCC |
In vitro transcription/translation assays
In vitro translation assay was performed using the TnT Quick Coupled Transcription/Translation System (Promega), according to the manufacturer’s instructions. Reactions were carried out using, 1 mM transcend biotin-lysyl-tRNA. The translation products were then separated on 10% SDS-PAGE gels, transferred onto nitrocellulose membrane and visualized by binding of streptavidin–horseradish peroxidase, followed by chemiluminescent detection. The assay was carried out on by pcDNA3.1(+) vector containing the full length of Linc00337 (V2), HOTAIR and the CDS sequences of MYC. HOTAIR functions a negative control, MYC as positive control.
Western blot
Cells were seeded into 6 cm plates (4 × 105 cells/plate) for 48 h and the lysates were subjected to SDS-PAGE electrophoresis, transferred onto a nitrocellulose membrane, and blocked with 5% skim milk at room temperature. The membranes were incubated with primary antibodies overnight, washed with phosphate buffered saline (PBS), and incubated with anti-mouse (Abcam, ab6728) or rabbit IgG-HRP (Abcam, ab6721). The primary antibodies were as follows: E2F1 (Abcam, ab218527), AURKB (Abcam, ab24), DSN1 (Proteintech, 17,742–1-AP), CCNB2 (Abcam, ab185622), CHEK1 (Abcam, ab40866), CENPA (Abcam, ab13939), DP1 (Abcam, ab124678), β-actin (Santa Cruz Biotechnology, sc69879), and GAPDH (Proteintech, 10,494–1-AP).
CCK8 cell viability assays
Approximately 2000 cells were seeded into 96-well plates and their absorption was measured every 24 h at 450 nm using a CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions.
Cells (500 cells/3 mL) were seeded into 12-well plates and incubated for 8–12 days at 37 °C with 5% CO2, with the medium changed every other day. Colonies were washed once with PBS, fixed with 0.5% paraformaldehyde for 20 min, and stained with crystal violet before the number of clones was counted. All assays were conducted in triplicate and results were expressed as the mean value.
Cell cycle analysis
The indicated cells were washed with 1× PBS, trypsinized, and collected before being fixed with 70% pre-coated ethanol at − 20 °C until further examination. Prior to analysis, samples were treated with 20 mg/mL of RNase (Sigma-Aldrich) for 1 h at 37 °C, labeled with 20 mg/mL propidium iodide (Sigma-Aldrich), and assessed by FACS Calibur flow cytometry (BD).
RNA-FISH assays
To detect the subcellular distribution of Linc00337, RNA-FISH assays were conducted. Briefly, cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 for 5 min on ice, and then incubated with RNA-FISH probes (RiboBio) in hybridization buffer at 37 °C overnight. Nuclei were counterstained with DAPI.
Dual luciferase reporter assays
To determine the effect of E2F1 on the Linc00337 promoter, stable E2F1-knockdown PANC1 cells and AsPC0-overexpressing cells were transfected with the PGL3- 337BS1/337BS2 construct and a Renilla luciferase reporter plasmid. After 24 h, firefly and Renilla luciferase activity were measured using a Dual Luciferase Reporter Assay System (Promega). To evaluate the effect of Linc00337 on E2F1 transcriptional activity, the cells were co-transfected with an E2F or E2F (mut) luciferase reporter plasmid (YEASEN) and Renilla plasmid. After 24 h, firefly and Renilla luciferase activity were measured using the Dual Luciferase Reporter Assay System (Promega).
RNA pull-down assay
Biotin-labeled RNA pull-down assays were performed as described previously. Briefly, Linc00337 and linc00337-AS were transcribed in vitro from linearized constructs using a Biotin-RNA Transcription Kit (Roche) and purified with TRIzol reagent (Invitrogen) before being incubated in RNA structure buffer (10 mM Tris-HCl pH 7, 0.1 M KCl, 10 mM MgCl2) and heated to 72 °C for 2 min to form proper secondary structures. The RNAs were then incubated with PANC1 cell lysates at 4 °C for 4 h followed by streptavidin beads (Thermo) at room temperature for 1 h, washed five times, and analyzed by western blot.
RNA immunoprecipitation assay (RIP)
RIPA was performed as described previously. Briefly, 2 × 107 PANC1 cells were crosslinked with 0.3% formaldehyde in medium for 10 min at room temperature, neutralized with 0 mM glycine for 5 min, and washed twice with cold PBS. Next, the cells were lysed in RIPA buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP-40, 0.5% sodium deoxycholate, 0.5 mM dithiothreitol, RNase and protease inhibitor cocktail), sonicated on ice, and treated with DNase before being pre-cleared with protein A/G beads (Thermo) for 30 min and incubated with Flag antibodies (Sigma, F3165) or mouse IgG (Abcam, ab190475) at 4 °C overnight. Antibodies were precipitated by incubation with protein A/G beads, washed five times for 10 min, and then RNA was extracted with Trizol reagent (Invitrogen) and detected by qRT–PCR. Protein samples were then subjected to western blotting.
Stably-transfected cells (5 × 106 cells) were subcutaneously injected into both flanks of at least five 6-week-old female BALB/c nude mice (Linchang Biotech). After 24 days, the mice were sacrificed and imaged. Animal care and experiments were performed in strict accordance with the “Guide for the Care and Use of Laboratory Animals” and were approved by the Committee for the Humane Treatment of Animals at Shanghai University of Medicine & Health Sciences. And the volume of tumor was calculated by the formula: L × W2 (L: the longest diameter of the tumour, W: the shortest diameter of the tumour).
Immunohistochemical (IHC) staining
Tumor slides were stained according to standard IHC protocols. Briefly, slides were blocked with 10% bovine serum albumin (Sangon) for 1 h, incubated with PCNA antibodies (Abcam, ab18197) overnight at 4 °C, and then incubated with HRP-labeled secondary antibodies (Dako) for 1 h at 25 °C. Antibodies were detected using diaminobenzidine substrate chromogen (DAB). All slides were counterstained with hematoxylin before dehydration and mounting.
Chromatin immunoprecipitation (ChIP) assays
ChIP assays were performed using a Pierce Agarose ChIP Kit according to the manufacturer’s protocol. PANC1 cells were crosslinked with 1% formaldehyde for 10 min at 37 °C and then incubated with anti-E2F1 antibodies (Abcam, ab4070) and anti-Rabbit IgG (Abcam, ab2410). Bound DNA fragments were subjected to RT-PCR using specific primers (Table
2).
Table 2
Correlations between Linc00337 and key clinicopathological parameters
Age | ≤50 years | 1 | 8 | 0.527 |
> 50 years | 2 | 7 |
Gender | Female2 | 0 | 3 | 0.396 |
Male1 | 3 | 12 |
Grade | I | 2 | 0 | 0.003* |
II and III | 1 | 15 |
Tumor size | ≤ 2 cm | 2 | 2 | 0.043* |
> 2 cm | 1 | 13 |
TNM stage | T1 | 1 | 0 | 0.05* |
T2 | 2 | 10 |
T3-T4 | 0 | 5 |
Chromatin isolation by RNA purification (ChIRP)
ChIRP was performed using PANC1 stable cell lines by adapting previously described protocols [
23] with minor modifications. Briefly, PANC1 cells were crosslinked with 1% glutaraldehyde for 10 min at room temperature with gentle shaking. Crosslinking was stopped with 0.25 mM glycine for 5 min. Cross-linked chromatin was incubated with biotinylated 20-mer antisense DNA probes targeting Linc00337 and negative control lacZ RNA (RiboBio) before streptavidin magnetic bead capture and wash/elution steps were performed as described previously [
23]. Eluted chromatin and RNA fragments were analyzed by qRT-PCR using the primers listed in Table
1.
Statistical analysis
Results were expressed as the mean ± standard deviation (SD) of at least three independent experiments. Between-group comparisons were analyzed by Student’s t-tests or analysis of variance. P values of < 0.05 were considered statistically significant.
Discussion
In this study, we reported Linc00337 not only overexpression in patients with PDAC but also closely corelated with clinicopathological parameters and further demonstrated that Linc00337 accelerates PDAC cell cycle transition and cell proliferation in vitro and tumor growth in vivo using loss- and gain-function assays. Moreover, increased Linc00337 expression correlated with poor clinical outcomes including OS and DFS, supporting our conclusion that Linc00337 exerts strong effects on PDAC cell proliferation and tumor growth.
LncRNAs play intricate roles via various different mechanisms, including the regulation of gene expression at the transcriptional and post-transcriptional levels [
30‐
32], with increasing evidence suggesting that lncRNA deregulation is associated with tumorigenesis and progression. Here, we demonstrated that Linc00337 exerts oncogenic effects by regulating the expression of E2F1, a crucial oncogenic transcription factor that plays key roles in cell cycle progression and cell proliferation in various malignancies [
9,
33]. Previous reports have identified several E2F1-associated lncRNAs, of which lncRNA-TINCR [
34], lncRNA-KHPS1 [
15], and Linc00668 [
17] are direct targets of E2F1 and lncRNA-GASL1 [
35] and lncRNA-H19 [
36] are related to its activity. These E2F1-related lncRNAs have been reported to play extensive roles in different malignant tumors; however, the underlying mechanisms are poorly understood. In this study, we verified that E2F1 directly binds to the promoter of Linc00337 and triggers its expression. We noticed that Linc00337 regulates a lot of genes involved in the late cell cycle phase including KIFs and CDCs. While, PDAC cells stopping in G1 phase after LINC0037 silencing, we supposed that the G1/S arrest in PDAC cells after Linc00337 silencing mainly due to the inhibition of E2F1, as the roles of E2Fs are pivotal in G1/S transition in human cells.
Mechanistically, we demonstrated that Linc00337 acts as an E2F1 co-activator by interacting with E2F1 and regulates its transcriptional activity. Since this interaction facilitates the activation of the E2F1 transcriptional network, a Linc00337-dependent E2F1 response may act via a positive feedback mechanism wherein E2F1 induces Linc00337 expression which promotes E2F1 expression and enhances its activity. However, future studies should investigate how Linc00337 regulates E2F1 expression.
Linc00337 overexpression has been previously reported in lung [
37] and gastric cancer tissues [
19]. In gastric and lung cancer Linc00337 seems to decrease the transcription of target genes and bind to DNMT1 [
21] or EZH2 [
19]. Here we are showing a transcriptional upregulation of target genes by Linc00337. We showed that Linc00337 promotes PDAC progression by enhancing the transcriptional activity of E2F1. All of these findings illustrate the oncogenic roles of Linc00337 in different cancers and we can’t rule out the same mechanisms which observed in gastric and lung cancer could be found in PDAC. We noticed that recently Yang et al. reported that Linc00337 recruits E2F4 to up-regulate TPX2 and induces autophagy and chemoresistance to cisplatin in esophageal squamous cell carcinoma [
22]. Their findings revealed that Linc00337 binds to E2F4, the other E2Fs family member [
38], taken our results and their findings together, Linc00337 seems to be largely related to the E2Fs family members and it should be further studied in the further. Moreover, we also found that Linc00337 is also involved in PDAC cell chemoresistance and metastasis both in vitro and in vivo (data will be published in the future). Taken together, Linc00337 exerts its oncogenic effects in different aspects in PDAC and other cancers by multiple mechanisms which indicates Linc00337 might be a key regulator in tumorigenesis and cancer progression. Therefore, our findings add another level of complexity to Linc00337-mediated gene regulation in different cancers.
Previous studies have indicated that lncRNAs display potential advantages in cancer diagnosis and prognosis [
39,
40]. In this study, we found that high Linc00337 levels were strongly associated with poor OS and DFS in PDAC patients; therefore, Linc00337 should be investigated further as an independent risk factor for PDAC patient survival.
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