- Email: [email protected]
RETRACTED: Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma progression by stimulating miR-874-3p and inhibiting PLK1 expression
Accepted Manuscript Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma progression by stimulating miR-874-3p and inhibiting PLK1 expression Wen-Jie Huang, Yun-Chao Wang, Song-Song Liu, Jia-Li Yang, Shi-Xiang Guo, LiJiang Wang, Huai-Zhi Wang, Ying-Fang Fan PII:
S0304-3835(18)30141-1
DOI:
10.1016/j.canlet.2018.02.014
Reference:
CAN 13764
To appear in:
Cancer Letters
Received Date: 4 January 2018 Revised Date:
4 February 2018
Accepted Date: 8 February 2018
Please cite this article as: W.-J. Huang, Y.-C. Wang, S.-S. Liu, J.-L. Yang, S.-X. Guo, L.-J. Wang, H.-Z. Wang, Y.-F. Fan, Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma progression by stimulating miR-874-3p and inhibiting PLK1 expression, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.02.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma ACCEPTED MANUSCRIPT progression by stimulating miR-874-3p and inhibiting PLK1 expression Wen-Jie Huanga, b,1, Yun-Chao Wangb, Song-Song Liub, Jia-Li Yangb, Shi-Xiang Guob, Li-Jiang Wangc, Huai-Zhi Wangb *, Ying-Fang Fana*
a.
Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University / The Second School of Clinical Medicine, Southern
b.
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Medical University, Industrial Road No.253, Guangzhou, Guangdong 510280, China
Institute of Hepatopancreatobiliary, Surgery Southwest Hospital, Third Military Medical University Third Military Medical University
(Army medical university), 30 Gaotanyan Main Street, Shapingba District Chongqing 400038, China c.
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Gulliver Preparatory School,6575 North Kendall Drive, Miami, Florida, 33156, U.S.
Corresponding authors:
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Huai-Zhi Wang, Institute of Hepatopancreatobiliary Surgery Southwest Hospital, Third Military Medical University Third Military Medical University (Army medical university), 30 Gaotanyan Main Street, Shapingba District, Chongqing, 400038. China. Email: [email protected], Tel:86-23-68765148.
Ying-Fang Fan, Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University / The Second School of Clinical Medicine,
Southern
Medical
University,
Industrial
Road
No.253,
Guangzhou,
Guangdong,
China.
Email:
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[email protected], Tel:86-13189097816.
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Abbreviations: CircRNAs, Circular RNAs; RBP, RNA-binding protein; miRNA, microRNA; PLK1, Polo like kinase 1; PDAC, pancreatic ductal adenocarcinoma; ceRNAs, Competing endogenous RNAs; siRNA, small interfering RNAs
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Abstract Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that regulate target gene expression by interacting with microRNA (miRNA). Emerging evidence shows that dysregulation of circRNAs plays important roles in biological and pathological processes, including cancer development and progression. The functional role of circRNA in PDAC (pancreatic ductal adenocarcinoma) remains to be investigated. In this study, high throughput microarray assay
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revealed that hsa_circ_0000977 was aberrantly up-regulated in pancreatic cancer tissues; this was also validated by qRT-PCR. Silencing hsa_circ_0000977 suppressed pancreatic cancer cell proliferation and induced cell cycle arrest, which was simulated by hsa-miR-874-3p mimics and blocked by hsa-miR-874-3p inhibitor. Bioinformatics analysis predicted that
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there is an hsa_circ_0000977/ hsa-miR-874-3p/ PLK1 (Polo like kinase 1) axis in pancreatic cancer progression. Dual-luciferase reporter system and FISH assay validated the direct interaction of hsa_circ_0000977, hsa-miR-874-3p, and
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PLK1. Western blot verified that inhibition of hsa_circ_0000977 decreased PLK1 expression. Furthermore, silencing hsa_circ_0000977 suppressed pancreatic cancer growth in vivo. Altogether, silencing hsa_circ_0000977 suppresses progression of pancreatic cancer by interacting with hsa-miR-874-3p and decreasing inhibiting PLK1 expression. Our results may provide a promising strategy for future diagnosis and treatment of pancreatic cancer.
Introduction
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Keywords: hsa_circ_0000977; miR-874-3p; ceRNAs; PLK1; PDAC proliferation.
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Pancreatic cancer is often asymptomatic in early stage and is one of the most common malignant tumors. In recent years, the incidence and mortality of pancreatic cancer have been increasing, particularly in younger patients[1]. Surgical
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resection is considered the best treatment choice for pancreatic cancer, but only 13%-20% patients have the opportunity to undergo surgery, and the 5-year survival rate is less than 5% after surgery [2, 3]. Various chemotherapeutic drugs, including gemcitabine, are regarded as a vital therapeutic strategy for pancreatic cancer patients after surgery, and can significantly improve survival rate. However, the therapeutic efficacy is often hampered by drug resistance [4]. Therefore, novel diagnostic biomarkers and therapeutic targets are urgently needed to improve prognosis for pancreatic cancer. Competing endogenous RNAs (ceRNAs) play important roles in post-transcriptional regulation. Disruption of the ceRNA networks is implicated in tumorigenicity [5-7]. circRNAs are also an important class of ceRNAs, which are endogenous, abundant, and stable in mammalian cells. One of the main roles of circRNAs is binding functional miRNAs to regulate gene expression [8, 9]. miRNAs are a class of widespread, small noncoding RNAs, which can negatively regulate their target gene expression by binding to the 3′-untranslated region of target mRNAs. Compared to circRNAs, miRNAs have been widely studied, and aberrant expression of miRNA is known to be associated with cancer cell proliferation,
apoptosis, invasion, and resistance to therapy [10]. Previous studies have demonstrated that miR-874-3p functioned as a
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critical mediator that could be a potential therapeutic target for hepatic cellular carcinoma (HCC) [11]. Other studies showed that miR-506 and miR-15a reduced proliferation and enhanced chemosensitivity of pancreatic cancer [12, 13]. It has been reported that the interaction of circRNAs and miRNAs are involved in some cancers, such as colorectal cancer, esophageal squamous cell carcinoma (ESCC) and HCC [14-16]. A previous study has also demonstrated that certain circRNAs were
development of pancreatic cancer remains to be elucidated.
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aberrantly expressed in pancreatic cancer [17], but the detailed functional role and mechanism of circRNAs in the
Polo like kinase 1 (PLK1) is a common serine/threonine protein kinase in the eukaryote that mediates various mitotic events, and is considered as a master cell cycle regulator [18]. Its role in regulating cell division, maintaining genome stability in mitosis, spindle assembly, and DNA damage response has been well recognized [19]. PLK1 is a key regulator of mitosis initiation, and
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abnormal PLK1 expression leads to dysregulation of the cell cycle, which is a main event in cancer initiation [20]. It has been confirmed that overexpression of PLK1 is associated with poor prognosis in most human cancers, and therefore targeting PLK1
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has been proposed to be a therapeutic strategy [21-23]. For example, small interfering RNAs (siRNAs) were used to repress tumor progression by inhibiting PLK1, as in the case of PLK1 inhibitors such as volasertib (BI6727) [24]. The carcinogenic effects of PLK1 in PDAC were also widely noted. Previous studies have demonstrated that PLK1 is closely associated with the PI3K/Akt pathway and plays important roles in cell proliferation and apoptosis in pancreatic carcinoma [25]. PLK1 inhibitors improved the anti-cancer efficacy of gemcitabine in pancreatic cancer [26, 27]. It was reported that
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siRNA-coupled superparamagnetic iron oxide nanoparticles or microRNA3686 can inhibit the expression of PLK1 and serve as therapeutic targets for PDAC [28, 29]. The overexpression of PLK1 is an adverse prognostic factor in various tumors and its expression is regulated by multiple factors. However, the interaction of circRNAs and PLK1 in PDAC
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remains to be investigated.
In this study, we found that hsa_circ_0000977 and PLK1 were aberrantly overexpressed in pancreatic cancer tissues.
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The aberrant expressions were closely associated with negative clinical characteristics and poor prognosis. Silencing hsa_circ_0000977 suppressed pancreatic cancer cell growth both in vitro and in vivo. We further demonstrated that hsa_circ_0000977/miR-874-3P/PLK1 axis plays an important role in regulating pancreatic cancer cell proliferation. Therefore, hsa_circ_0000977 overexpression may serve as a promising biomarker for early diagnosis and prognosis of PDAC and silencing hsa_circ_0000977 could be a therapeutic strategy.
Materials and methods Chemicals The DAB reagent kit was obtained from Boster Biological Technology Co. Ltd (Wuhan, China). Phosphate-buffered saline (PBS) was obtained from Wuhan Boster Biological Technology Co. Ltd. RIPA Lysis Buffer was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Lipofectamine 2000 was obtained from Invitrogen (Camarillo, CA,
USA). The Cell Counting Kit-8 was obtained from Dojinodo (Shanghai, China). Enhanced BCA Protein Assay Kit was
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obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Propidium iodide was obtained from Kaiji (Nanjing, China). Chemiluminescent HRP Substrate was obtained from Millipore (Billerica, MA, USA). TRIzol Reagent was obtained from Invitrogen. Prime Script RT reagent Kit, Mir-X miR First-Strand Synthesis Kit, and SYBR Premix Ex Taq II were obtained from TaKaRa (Dalian, China). The anti-PLK1, anti-cyclin D1, anti-bcl-2, anti-cleaved caspase-3
control vector were purchased from GeneChem (Shanghai, China). Cell culture
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monoclonal antibody (mAb) was obtained from Abcam (Shanghai, China). Lentiviral-hsa_circ_0000977-RNAi and the
Two pancreatic cell lines, AsPC-1 and PANC-1 (Chinese Academy of Sciences), were used in this study. The two cell lines were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% antibiotics and maintained at 37°C in a
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humidified atmosphere with 5% CO2. Pancreatic cancer tissue specimens
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Fifty matched cancer and adjacent non-cancerous tissues, seventy-one cancer tissues, and five normal tissues were used in this study. Fifty matched cancer and adjacent non-cancerous tissues, fifty-one cancer tissues, and five normal tissues were obtained from the Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University. The other twenty cancer tissues were obtained from the Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern
specimens. Analyzing circRNA expression profile
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Medical University. The ethics committee of both ZhuJiang Hospital and Southwest Hospital approved the use of clinical
Twenty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues were analyzed using the
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circRNAs chips. The microarray hybridization and collection of data were performed by KangChen Bio-tech, Shanghai, China. The five most up- and down-regulated circRNAs and their hierarchical clustering analysis were performed based on
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their expression value using the Cluster and TreeView program. RNA extraction and qRT-PCR
Thirty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and eighteen cancer tissues were used to perform qRT-PCR. TRIzol Reagent (Invitrogen) was used to extract total RNA from these tissues. For circRNAs, RNase R was used to degrade linear RNA, which have poly (A), and amplified by divergent primer. qRT-PCR analysis on circular RNA and mRNA was performed using Prime Script RT reagent Kit (TaKaRa) and SYBR Premix Ex Taq II (TaKaRa). β-actin was used as an endogenous control. For miR-874-3P analysis, miRNA was treated with DNase I to eliminate genomic DNA and cDNA was synthesized by Mir-X miR First-Strand Synthesis Kit (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for qRT-PCR. The expression was normalized to RNU6-2. The 2-∆∆CT method was adopted to calculate relative expression of hsa_circ_0000977 and hsa-miR-874-3p. Cell transfection
AsPC-1 and PANC-1 cells were seeded in 6-well plates and cultured to 60-70% confluence before transfection.
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According to the manufacturer’s instructions, miRNA mimics, inhibitors (Sangon, China), and siRNA (GeneChem, China) were transiently transfected using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM. The corresponding negative controls were transfected simultaneously under the same condition. Lentiviral-hsa_circ_0000977-RNAi and the negative control lentiviruses were purchased from GeneChem (Shanghai, China) and transfected according to the manufacturer’s protocol.
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Protein isolation and Western blot
Twelve matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and additional five normal tissues from above (qRT-PCR) were lysed using RIPA buffer according to manufacturer's instructions. 8% SDS-PAGE was used to separate protein extracts. After electrophoresis, protein extracts were transferred onto Nitrocellulose membranes
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(Biosharp) in 200 mA for 2 h, followed by blocking for another 2 h, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies for 2 h at room temperature. HRP activity was detected using Chemiluminescent
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HRP Substrate (Millipore, Billerica, MA, USA) and visualized with a UVP BioImaging system. The protein isolation and Western blot analysis on transfected pancreatic cells were performed following the same procedures. Cell proliferation assay
After transfection, cells were seeded in 96-well plates with 3000 cells per well. 10 µl of the Cell Counting Kit-8 reagent (Dojinodo, Shanghai, China) was added to each well and the OD value was measured after 4 h incubation. We
Cell cycle assay
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record the OD value of 24, 48, 72, and 96 h after transfection for statistical analysis.
For cell cycle analysis, transfected AsPC-1 and PANC-1 cells were fixed in 70% ethanol overnight at -20°C and
Colony formation assay
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stained with propidium iodide (Kaiji, Nanjing, China). Cell cycle assays were conducted at 48 h after transfection.
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One hundred transfected pancreatic cancer cells were seeded in 6-well plates. After 2 weeks of incubation, colonies (>200 cells per colony) were stained with Giemsa. The number of colonies was counted. Each experiment was performed in triplicate.
Luciferase reporter assay
HEK293T cells were seeded in 96-well plates and cultured to 50-70% confluence before transfection. The cells were divided into four groups: PLK1-3'UTR and hsa-miR-874-3p, PLK1-3'UTR and N.C, PLK1-3'UTR-muta and hsa-miR-874-3p, PLK1-3'UTR-muta and N.C. For every corresponding experimental group, 0.16 µg plasmids of PLK13’UTR and PLK13’UTR-muta, 5 pmol of hsa-miR-874-3p and N.C. were used. After 48 h incubation, Promega Dual-Luciferase system was used to detect firefly and Renilla luciferase activities. Using 100 µl Luciferase Assay Reagent II (LAR II) (Luciferase Assay Reagent, Progema) and subsequently 20 µl lysis buffer, firefly luciferase activities were measured as internal reference, and Renilla luciferase activities were also measured using 100 µl Stop & Glo® Reagent
(Luciferase Assay Reagent, Progema). Finally, the subtracted difference of firefly and Renilla luciferase activities were
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calculated as relative luciferase activity. Immunohistochemistry (IHC) Thirty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and additional fifty-three cancer tissues were used for paraffin embedding and sectioning. IHC analysis was performed under manufacturer’s instructions. Briefly, the slides were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies at
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room temperature for 2 h. The expression was evaluated using a composite score obtained by multiplying the values of staining intensities (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage of positive cells (0, 0%; 1, <10%; 2, 10-50%; 3, > 50%). Fluorescence in situ hybridization (FISH)
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The hybridization was performed overnight with hsa_circ_0000977 and hsa-miR-874-3p probes. Specimens were analyzed on a Nikon inverted fluorescence microscope. The hsa_circ_0000977 probe for FISH was 5’-TGGAT GTTGT
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TTACT GATTC TGGCA ATTAA-3’ and the miR-874-3p probe for FISH was 5’-TCGGT CCCTC GGGCC AGGGC AG-3’. Xenografts experiments
Five-week-old female BALB/c nude mice were selected for this study. A total number of 2×106 AsPC-1 cells transfected with Lentiviral-hsa_circ_0000977-RNAi and negative control were subcutaneously injected into the back of nude mice. The
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volume and weight of tumors were measured after 20 days. The Institutional Animal Care and Use Committee of the Third Military Medical University Animal approved the experimental procedures. Statistical analysis
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Statistical analysis was accomplished using SPSS 22.0 (version 22.0, Chicago, IL, USA). The data was presented as means ± s.d. For quantitative data, Student’s t test and Mann-Whitney U test were used. For categorical data, Chi-square test was used. The
when P < 0.05.
RESULTS
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log-rank test was used to compare different survival curves by Kaplan-Meier analysis. Significant difference was considered
Informatics reveal circRNA hsa_circ_0000977/hsa-miR-874-3p/PLK1 axis Using high throughput microarray assay, we identified the expression profiles of dysregulated circRNAs. We found 289 circRNAs were aberrantly expressed with fold change ≥ 2.0 and P < 0.05. Among them, 128 circRNAs were up-regulated and 161 circRNAs were down-regulated. Fold change filtering displayed the differentially expressed circRNAs (Fig. 1A) and Volcano Plot Filtering identified differentially changed circRNAs with statistical difference between carcinoma and non-carcinoma tissues (Fig. 1B). Hierarchical clustering showed the five most up- and down-regulated
circRNAs between cancer and adjacent non-cancerous tissues (Fig. 1C). Then, we further confirmed the expression of these
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5 most up-regulated circRNAs in 5 matched PDAC and non-tumor specimens, and found that hsa_circ_0000977 was consistently and significantly increased in PDAC tissues as compared to matched controls by nearly 71 folds in the microarray assays (Supplementary Fig. 1). To investigate the potential miRNAs associated with hsa_circ_0000977, TargetScan and miRanda database were used, and the five most potentially complementary binding miRNAs were presented (Supplementary Table 1). MiRWalk 2.0, Gene ontology (GO) and KEGG enrichment analysis were used to investigate the
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potential target of miRNAs (Fig. 1D). Among these target miRNAs, we found that miR-874-3p has the potential to interact with PLK1. The microRNA response elements (MREs) are shown in Fig. 1E. These results suggested that hsa_circ_0000977 may have the potential to regulate the expression of PLK1 by sequestering miR-874-3p, in turn promoting the progression of pancreatic cancer.
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Overexpression of hsa_circ_0000977 is correlated with poor prognosis in PDAC patients
We examined the expression of hsa_circ_0000977 and miR-874-3p by qRT-PCR in 30 matched PDAC and adjacent
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non-cancer tissues. Gel electrophoresis and DNA sequence confirmed the PCR product of hsa_circ_0000977 (Fig. 2A). qRT-PCR showed higher expression of hsa-circ-0000977 and lower expression of miR-874-3p in cancer tissues than in matched non-tumor tissues (Fig. 2B, 2C). In pancreatic cancer tissues, hsa_circ_0000977 showed higher expression than miR-874-3p (Supplementary Fig.2). This was verified in the same batch of tissues, indicating that the expression of hsa-circ-0000977 and miR-874-3p had a negative correlation. Furthermore, the expression of hsa_circ_0000977 in 48
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pancreatic cancer tissues was examined. Based on the median value of expression, patients were classified into low and high expression groups. Compared to the low expression group, Kaplan-Meier survival curves showed that the patients with high expression of hsa_circ_0000977 had a low 5-year overall survival rate (12 VS 16.5 months, P=0.0403) (Fig. 2D). These
PDAC patients.
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results demonstrate that hsa_circ_0000977 is overexpressed in PDAC tissues and is associated with poor prognosis of
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hsa_circ_0000977 regulates cell proliferation and cell cycle by sequestering miR-874-3P in vitro The roles of hsa_circ_0000977 in PADC progression were further investigated. To block the hsa_circ_0000977 expression, two pancreatic cell lines, AsPC-1 and PANC-1, were transfected with hsa_circ_0000977 siRNA and the silencing efficiency was confirmed by qRT-PCR. After transfection, the expression of hsa_circ_0000977 and PLK1 decreased compared to the controls (Supplementary Fig. 3). Since PLK1 is involved in the initiation, maintenance, and completion of cell mitosis, and has interaction with hsa_circ_0000977, we assumed that hsa_circ_0000977 might also be involved in cell cycle regulation and cell proliferation. CCK-8 assay showed that hsa_circ_0000977 siRNA and miR-874-3P mimics induced a proliferation-suppressing effect, whereas the suppressing effect was reversed by an miR-874-3P inhibitor (Fig. 3A). Consistently, colony formation assays showed that hsa_circ_0000977 siRNA and miR-874-3P mimics decreased colony formation ability, and no significant difference was observed when hsa_circ_0000977 siRNA and miR-874-3P inhibitor were transfected simultaneously (Fig. 3B). Flow cytometry demonstrated that hsa_circ_0000977 siRNA and
miR-874-3P mimics induced G1/S arrest, and co-transfection of hsa_circ_0000977 siRNA and miR-874-3P inhibitor
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abrogated the cell cycle block effects (Fig. 3C). The above assays were also repeated in PANC-1 cell lines, which showed similar results (Supplementary Fig. 4). These results suggest that hsa_circ_0000977 modulates proliferation and the cell cycle as a miR-874-3P sponge in PDAC cells. hsa_circ_0000977 regulates PLK1 by sequestering miR-874-3p Bioinformatics analysis showed that both hsa_circ_0000977 and PLK1 contain mutual miRNA response elements (MREs)
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to miR-874-3p. We hypothesized that hsa_circ_0000977 exerts its biological function as a miR-874-3p sponge to regulate PLK1 expression. Dual-luciferase reporter assays were then performed to confirm whether there was a direct interaction between hsa_circ_0000977 and miR-874-3p, or between miR-874-3p and PLK1. The alignments of potential binding sites and their mutant types were constructed through bioinformatics analysis (Fig. 4A, 4B). There was significant reduction of
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firefly luciferase reporter activity for transfected hsa-miR-874-3p and hsa-circ-0000977 WT, and there was no notable change of luciferase reporter activity in the hsa-circ-0000977 mutant (Fig. 4C). Co-transfection of hsa-miR-874-3p and PLK1 WT also
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significantly decreased the firefly luciferase reporter activity (Fig. 4D). These results indicated that direct binding interactions existed between hsa_circ_0000977 and miR-874-3p, and between miR-874-3p and PLK1. qRT–PCR was used to investigate whether hsa-circ-0000977 regulates PLK1mRNA expression via hsa-miR-874-3p. Both hsa_circ_0000977 siRNA and miR-874-3p mimics significantly reduced the PLK1 mRNA expression in pancreatic cancer cells. There was no notable change in PLK1 expression when cells were simultaneously treated with an hsa_circ_0000977 siRNA and a
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miR-874-3p inhibitor, indicating that a miR-874-3p inhibitor could significantly rescue the decrease of PLK1 induced by silencing hsa_circ_0000977 (Fig. 4E, 4F). The interaction between hsa_circ_0000977 and miR-874-3p was further investigated by FISH analysis in AsPC-1 and PANC-1 cell lines. We found that hsa_circ_0000977 and miR-874-3p were
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co-localized in the cytoplasm, which suggested that hsa_circ_0000977 binds to miR-874-3p (Fig. 4G). Taken together, these results confirm our hypothesis that hsa_circ_0000977 regulates PLK1 through miR-874-3p in pancreatic cancer
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progression.
PLK1 is overexpressed in pancreatic cancer tissues and regulated by hsa_circ_0000977 Based on computational algorithms and clinical application significance, we chose PLK1 as the presumed target of hsa_circ_0000977/miR-874-3P. Twelve pairs of matched pancreatic cancer tissues and adjacent non-cancerous tissues, and 5 normal tissues were examined for the expression of PLK1. Through Western blot analysis, we found that PLK1 expression was significantly up-regulated in cancer tissues (Fig. 5A). To further investigate PLK1 expression in the tissues, we performed immunohistochemistry in 30 matched tissues. We found that PLK1 showed high expression in pancreatic cancer tissues in which hsa_circ_0000977 was also overexpressed (Fig. 5B). Furthermore, we found that PLK1 was up-regulated in 83 archival formalin-fixed, paraffin-embedded pancreatic cancer tissues, especially in advanced stage pancreatic cancer (Supplementary Fig. 5). The level of PLK1 expression was significantly associated with clinical stage, as patients with high PLK1 expression were more likely in advanced stage (Supplementary Table 2). Therefore, Western blot was performed to
examine whether hsa_circ_0000977 can affect PLK1 protein expression through miR-874-3P. In PDAC cell lines, both
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silencing of hsa_circ_0000977 or transfection with miR-874-3P mimic significantly reduced the expression of PLK1. PLK1 expression was rescued when miR-874-3p inhibitor was co-transfected with hsa_circ_0000977 siRNA (Fig. 5C). There were also signs that PLK1 could interact with other gene products, indicating that PLK1 may play an important role in other cancerous events such as apoptosis. The associated proteins were then examined. After knockdown of hsa_circ_0000977, cell cycle-related proteins cyclin D1 and bcl-2 were significantly decreased. However, apoptosis-related proteins cleaved
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caspase-3 were significantly increased (Fig. 5D). Finally, these results confirmed that overexpression of PLK1 was a significant feature of pancreatic cancer, and that hsa_circ_0000977 modulated PLK1 as a miR-874-3P sponge in PDAC. Knockdown of hsa_circ_0000977 inhibits pancreatic cell growth in vivo
Silencing hsa_circ_0000977 suppresses proliferation of pancreatic cancer cell in vitro. We investigated whether this is
with
low
hsa_circ_0000977
expression
via
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also true in vivo using a xenograft nude mouse model. AsPC-1 cells were used to establish a stable pancreatic cancer cell line Lentiviral-hsa_circ_0000977-RNAi.
The
biological
impact
of
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hsa_circ_0000977-RNAi was measured by CCK-8 assays and flow cytometry analysis. The results showed that Lentiviral-hsa_circ_0000977-RNAi significantly decreased hsa_circ_0000977 expressions, reduced cell proliferation and blocked cell cycle (Supplementary Fig. 6).
AsPC-1 cells with low or normal hsa_circ_0000977 expression induced by transfection with lentiviral or control vectors were subcutaneously implanted into nude mice. After 20 days, the weights of the tumors were measured. Tumors
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derived from low hsa_circ_0000977 expression group had lower weight than their negative controls (Fig. 6A). The expression level of PLK1 was examined by IHC. The results showed that, compared to negative controls, expression of PLK1 significantly decreased (Fig. 6B). These results demonstrate that hsa_circ_0000977 can suppress pancreatic cancer
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Discussion
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progression in vivo and decrease PLK1 expression.
CircRNAs are a novel class of extensive and stable endogenous RNAs that regulate gene expression in mammals [30-32]. The covalently closed loop structures render circRNAs more stable than liner RNA and insusceptible to RNA exonuclease or RNase [33]. CircRNAs mainly act as a miRNA sponge to exert their post-transcriptional functions as ceRNAs, which is more effective than the traditional anti-miRNA approach [8]. It has been elucidated that some circRNAs may be potential biomarkers for the diagnosis of gastric cancer, hepatocellular carcinoma, lung cancer, colon carcinoma, leukemia, and other cancers [34]. Because of the cell type or developmental stage specific characteristics, circRNAs may also play roles in PDAC progression and serve as valuable clinical biomarkers for PDAC. Compared to circRNAs, miRNAs have been well studied. Many studies have documented that dysregulation of miRNA is closely associated with tumorigenesis. miRNAs inhibit target protein translation by interacting with the untranslated region (3’-UTR) of target
mRNAs [35]. Previous studies have shown that miRNAs play diverse and significant roles in PDAC progression and
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metastasis [36]. In the ceRNA networks, circRNAs have strong inhibitory effect on miRNAs, and the interaction between circRNAs and miRNAs has already been observed to perform significant functions in a variety of cancers. In this study, hsa_circ_0000977 was found to be aberrantly up-regulated in pancreatic cancer tissues by high throughput microarray assay. This finding was further confirmed by qRT-PCR. Based on bioinformatics analysis, it was assumed that the hsa_circ_0000977/miR-874-3P/PLK1 axis plays a pivotal role in pancreatic cancer progression. Luciferase reporter system
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showed that miR-874-3P targets both hsa_circ_0000977 and PLK1, which provided direct evidence that hsa_circ_0000977 functions as a miR-874-3P sponge to modulate PLK1 expression. FISH analysis demonstrated that hsa_circ_0000977 and miR-874-3P primarily reside in the cytoplasm, and their co-location explained their post-transcriptional regulatory role. Cytological function experiment and flow cytometry revealed that hsa_circ_0000977 and miR-874-3P have reverse effects
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in cell phenotype and cell cycle. A miR-874-3P inhibitor could rescue biological changes induced by silencing of hsa_circ_0000977. Taken together, the study revealed that an hsa_circ_0000977/miR-874-3P/PLK1 axis exists in PDAC,
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and that hsa_circ_0000977 negatively regulates miR-874-3P. The aberrantly up-regulated hsa_circ_0000977 accompanied with by down-regulated miR-874-3P may be potentially used for early diagnosis and determining prognosis in pancreatic cancer patients.
PLK1 is overexpressed in various types of cancers, and dysregulation of PLK1 causes tumorigenesis by disrupting many events in cellular processes [37, 38]. Despite its important roles in the cell cycle, PLK1 also has mutual interactions
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with other pivotal molecules in cells [39]. Previous studies have shown that chemotherapy resistance, which is a common and early occurrence in pancreatic cancer, is often associated with PLK1 overexpression [40]. In this study, we confirmed that PLK1 was over-expressed in PDAC tissues and was related to histological grade and TNM stage. Hsa_circ_0000977
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promoted PDAC progression by up-regulating PLK1 expression through the hsa_circ_0000977/ miR-874-3P/PLK1 axis, and silencing of hsa_circ_0000977 inhibited PLK1 expression.
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In conclusion, this is the first study to investigate the regulatory function of hsa_circ_0000977 in PDAC and the interaction between hsa_circ_0000977, miRNA, and PLK1. Through the hsa_circ_0000977/miR-874-3P/PLK1 axis, hsa_circ_0000977 performed specific regulatory roles in the proliferation and progression of PDAC. Hsa_circ_0000977 could be a novel biomarker of poor prognosis in pancreatic cancer. In addition, the hsa_circ_0000977/miR-874-3P/PLK1 axis is a special signaling pathway, which could be a potential therapeutic target for pancreatic cancer patients.
Fundings This work was supported by the National Key R&D Program of China (No.2017YFC1308600); The National Key Research Program of China (2016YFC1201802); the National Nature Science Foundation of China (No.81672382, 81502550, 81371652); the Clinical Research Foundation of TMMU (No. SWH2015LC01, SWH2016JCZD01).
Acknowledgement： ：We thank Dr. Dong-Hua Yang(Fox Chase Cancer Center, Philadelphia) for helping to revise the
manuscript.
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Conflict of interests： ：The authors declare that they have no competing interests.
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Fig legends Fig. 1. The identification of circRNA expression profiles and prediction of hsa_circ_0000977 functional mechanism (A)Scatter plot of circRNAs expression in PDAC and adjacent non-carcinoma tissues. The circRNAs above the top green line and below the bottom green line are those up or down-regulated more than 2.0 folds. (B) Volcano plot visualizes the different
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expression in these two groups. The red dots represent more than 2.0 fold changes (log2 scaled) of circRNAs and with statistical significance (P <0.05). (C) Hierarchical cluster analysis of the top five up- and down-regulated circRNAs in 20 pairs of matched tissues. (D) The association of circRNA-miRNA-mRNAs in the five most up-regulated circRNAs. In this network, the interaction of the five most up-regulated circRNAs (green circle), 12 targeted miRNAs (orange circle) and 207
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microRNA response elements (MREs) of hsa-miR-874-3p.
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targeted mRNAs (green triangle) were exhibited. (E) TargetScan and miRanda database confirm hsa_circ_0000977 contains
Fig. 2. The expression of hsa_circ_0000977 and miR-874-3p
(A) PCR product of hsa_circ_0000977 in 1.5% agarose gelelectrophoresis and DNA sequencing analysis. DL2000 DNA marker was used (from up to down, 2000,1000, 750, 500, 250, 100 bp). (B) qRT–PCR on Hsa_circ_0000977 expression in 30 paired pancreatic cancer tissues and adjacent non-cancer tissues. (C) qRT–PCR on miR-874-3p expression in 30 paired pancreatic cancer tissues and
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adjacent non-cancer tissues. (D) The Kaplan–Meier survival analysis and log-rank test show up-regulation of hsa_circ_0000977 correlates with low 5-year overall survival rates. The median survival time for patients with high and low expression of
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hsa_circ_0000977 was 12 months as compared to 16.5 months respectively (P = 0.0403).
Fig. 3. Silencing hsa_circ_0000977 suppresses pancreatic cancer cell proliferation and induces cell cycle arrest in AsPC-1 cell
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(A) CCK-8 assay. hsa_circ_0000977 siRNA and miR-874-3P mimics suppresses cancer cell proliferation, miR-874-3P inhibitor abolishes the suppression effect of has_circ-0000977 knockdown. OD values are obtained at 24,48,72,96 hours after transtection. (B) Colony formation assay. hsa_circ_0000977 siRNA and miR-874-3P mimics reduced cancer cell colony forming ability, miR-874-3P inhibitor restores the inhibiting effect of has_circ-0000977 siRNA. (C) Flow cytometric analysis. Cell block were observed when hsa_circ_0000977 siNRA and miR-874-3P mimics were transfected. Co-transfection of has_circ-0000977 siRNA and miR-874-3P inhibitor have no obvious impact on cell cycle. Experiments were repeated 3 times and data were presented as means±s.d. NC, negative control. *P<0.05, **P<0.01, ***P<0.001.
Fig.4. The interaction of hsa_circ_0000977, miR-874-3p and PLK1 (A) miRNA response elements (MREs) is showed by which hsa_circ_0000977 sequesters miR-874-3p. Mutations were
generated in MREs. (B) Putative miR-874-3p binding sites in the 3′-UTR of PLK1 mRNA is showed. The sequence in the
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PLK1 3′-UTR at the complementary sites of miR-874-3p were mutated. (C, D) Dual luciferase reporter show significant reduction of luciferase activity of the wild-type and luciferase activity is restored by the mutant sequence. Compared to mutant group, the luciferase activity of hsa_circ_0000977 wild type has decreased 23% and the luciferase activity of PLK1 wild type has decreased 30%. The relative luciferase activities were normalized with Renilla activity. (E, F) qRT-PCR illustrates inhibition of circ-0000977 and overexpression of miR-874-3p degrades PLK1 mRNA, and miR-874-3p inhibitor
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restores PLK1 mRNA expression. (G) The co-localization of hsa_circ_0000977 and miR-874-3p in pancreatic cancer cells by FISH. *P<0.05, **P<0.01, ***P<0.001.
Fig. 5. PLK1 overexpressed in pancreatic cancer tissues and hsa_circ_0000977 regulates PLK1 expression by acting
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as miR874-3p sponge
(A)Western blot analysis in 12 pairs of pancreatic cancer tissues show PLK1 is up-regulated in cancer tissues compared to
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adjacent noncancerous and normal tissues. Aj, adjacent non-cancerous tissue; T, pancreatic cancer; N, normal tissues. (B) Immunohistochemistry on PLK1 expression in pancreatic cancer and adjacent non-cancerous tissues. (C)Silencing of hsa_circ_0000977 by siRNA inhibiting PLK1expression，overexpression of miR874-3p by mimics suppress PLK1 expression and miR874-3p inhibitor restore the PLK1expression which is suppressed by hsa_circ_0000977 siRNA. (D)
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silencing of hsa_circ_0000977 increase caspase-3 expression and decrease cyclin D1and bcl-2 expression.
Fig. 6. Knockdown of hsa_circ_0000977 suppresses pancreatic cancer growth and PLK1 expression (A)Representative graph show tumor growth and the mean tumor weights 30 days after subcutaneously implanted AsPC-1
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cells. (B) Compared negative control, The PLK1 expression was decreased.
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ACCEPTED MANUSCRIPT Highlights: 1. Aberrantly up-regulated hsa_circ_0000977 play an important role in PDAC proliferation.
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2. hsa_circ_0000977 and miR-874-3P exert their post transcriptional regulation roles in PDAC.
3. hsa_circ_0000977 act as miR-874-3P sponge to exert its oncogenic roles.
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4. hsa_circ_0000977/ miR-874-3P/PLK1 axis is established and confirmed in PDAC
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5. Knockdown of hsa_circ_0000977 surely suppress PDAC growth by inhibiting
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PLK1 in vivo.
S0304-3835(18)30141-1
DOI:
10.1016/j.canlet.2018.02.014
Reference:
CAN 13764
To appear in:
Cancer Letters
Received Date: 4 January 2018 Revised Date:
4 February 2018
Accepted Date: 8 February 2018
Please cite this article as: W.-J. Huang, Y.-C. Wang, S.-S. Liu, J.-L. Yang, S.-X. Guo, L.-J. Wang, H.-Z. Wang, Y.-F. Fan, Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma progression by stimulating miR-874-3p and inhibiting PLK1 expression, Cancer Letters (2018), doi: 10.1016/j.canlet.2018.02.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Silencing circular RNA hsa_circ_0000977 suppresses pancreatic ductal adenocarcinoma ACCEPTED MANUSCRIPT progression by stimulating miR-874-3p and inhibiting PLK1 expression Wen-Jie Huanga, b,1, Yun-Chao Wangb, Song-Song Liub, Jia-Li Yangb, Shi-Xiang Guob, Li-Jiang Wangc, Huai-Zhi Wangb *, Ying-Fang Fana*
a.
Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University / The Second School of Clinical Medicine, Southern
b.
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Medical University, Industrial Road No.253, Guangzhou, Guangdong 510280, China
Institute of Hepatopancreatobiliary, Surgery Southwest Hospital, Third Military Medical University Third Military Medical University
(Army medical university), 30 Gaotanyan Main Street, Shapingba District Chongqing 400038, China c.
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Gulliver Preparatory School,6575 North Kendall Drive, Miami, Florida, 33156, U.S.
Corresponding authors:
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Huai-Zhi Wang, Institute of Hepatopancreatobiliary Surgery Southwest Hospital, Third Military Medical University Third Military Medical University (Army medical university), 30 Gaotanyan Main Street, Shapingba District, Chongqing, 400038. China. Email: [email protected], Tel:86-23-68765148.
Ying-Fang Fan, Hepatobiliary Surgery, Zhujiang Hospital, Southern Medical University / The Second School of Clinical Medicine,
Southern
Medical
University,
Industrial
Road
No.253,
Guangzhou,
Guangdong,
China.
Email:
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[email protected], Tel:86-13189097816.
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Abbreviations: CircRNAs, Circular RNAs; RBP, RNA-binding protein; miRNA, microRNA; PLK1, Polo like kinase 1; PDAC, pancreatic ductal adenocarcinoma; ceRNAs, Competing endogenous RNAs; siRNA, small interfering RNAs
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Abstract Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that regulate target gene expression by interacting with microRNA (miRNA). Emerging evidence shows that dysregulation of circRNAs plays important roles in biological and pathological processes, including cancer development and progression. The functional role of circRNA in PDAC (pancreatic ductal adenocarcinoma) remains to be investigated. In this study, high throughput microarray assay
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revealed that hsa_circ_0000977 was aberrantly up-regulated in pancreatic cancer tissues; this was also validated by qRT-PCR. Silencing hsa_circ_0000977 suppressed pancreatic cancer cell proliferation and induced cell cycle arrest, which was simulated by hsa-miR-874-3p mimics and blocked by hsa-miR-874-3p inhibitor. Bioinformatics analysis predicted that
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there is an hsa_circ_0000977/ hsa-miR-874-3p/ PLK1 (Polo like kinase 1) axis in pancreatic cancer progression. Dual-luciferase reporter system and FISH assay validated the direct interaction of hsa_circ_0000977, hsa-miR-874-3p, and
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PLK1. Western blot verified that inhibition of hsa_circ_0000977 decreased PLK1 expression. Furthermore, silencing hsa_circ_0000977 suppressed pancreatic cancer growth in vivo. Altogether, silencing hsa_circ_0000977 suppresses progression of pancreatic cancer by interacting with hsa-miR-874-3p and decreasing inhibiting PLK1 expression. Our results may provide a promising strategy for future diagnosis and treatment of pancreatic cancer.
Introduction
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Keywords: hsa_circ_0000977; miR-874-3p; ceRNAs; PLK1; PDAC proliferation.
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Pancreatic cancer is often asymptomatic in early stage and is one of the most common malignant tumors. In recent years, the incidence and mortality of pancreatic cancer have been increasing, particularly in younger patients[1]. Surgical
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resection is considered the best treatment choice for pancreatic cancer, but only 13%-20% patients have the opportunity to undergo surgery, and the 5-year survival rate is less than 5% after surgery [2, 3]. Various chemotherapeutic drugs, including gemcitabine, are regarded as a vital therapeutic strategy for pancreatic cancer patients after surgery, and can significantly improve survival rate. However, the therapeutic efficacy is often hampered by drug resistance [4]. Therefore, novel diagnostic biomarkers and therapeutic targets are urgently needed to improve prognosis for pancreatic cancer. Competing endogenous RNAs (ceRNAs) play important roles in post-transcriptional regulation. Disruption of the ceRNA networks is implicated in tumorigenicity [5-7]. circRNAs are also an important class of ceRNAs, which are endogenous, abundant, and stable in mammalian cells. One of the main roles of circRNAs is binding functional miRNAs to regulate gene expression [8, 9]. miRNAs are a class of widespread, small noncoding RNAs, which can negatively regulate their target gene expression by binding to the 3′-untranslated region of target mRNAs. Compared to circRNAs, miRNAs have been widely studied, and aberrant expression of miRNA is known to be associated with cancer cell proliferation,
apoptosis, invasion, and resistance to therapy [10]. Previous studies have demonstrated that miR-874-3p functioned as a
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critical mediator that could be a potential therapeutic target for hepatic cellular carcinoma (HCC) [11]. Other studies showed that miR-506 and miR-15a reduced proliferation and enhanced chemosensitivity of pancreatic cancer [12, 13]. It has been reported that the interaction of circRNAs and miRNAs are involved in some cancers, such as colorectal cancer, esophageal squamous cell carcinoma (ESCC) and HCC [14-16]. A previous study has also demonstrated that certain circRNAs were
development of pancreatic cancer remains to be elucidated.
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aberrantly expressed in pancreatic cancer [17], but the detailed functional role and mechanism of circRNAs in the
Polo like kinase 1 (PLK1) is a common serine/threonine protein kinase in the eukaryote that mediates various mitotic events, and is considered as a master cell cycle regulator [18]. Its role in regulating cell division, maintaining genome stability in mitosis, spindle assembly, and DNA damage response has been well recognized [19]. PLK1 is a key regulator of mitosis initiation, and
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abnormal PLK1 expression leads to dysregulation of the cell cycle, which is a main event in cancer initiation [20]. It has been confirmed that overexpression of PLK1 is associated with poor prognosis in most human cancers, and therefore targeting PLK1
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has been proposed to be a therapeutic strategy [21-23]. For example, small interfering RNAs (siRNAs) were used to repress tumor progression by inhibiting PLK1, as in the case of PLK1 inhibitors such as volasertib (BI6727) [24]. The carcinogenic effects of PLK1 in PDAC were also widely noted. Previous studies have demonstrated that PLK1 is closely associated with the PI3K/Akt pathway and plays important roles in cell proliferation and apoptosis in pancreatic carcinoma [25]. PLK1 inhibitors improved the anti-cancer efficacy of gemcitabine in pancreatic cancer [26, 27]. It was reported that
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siRNA-coupled superparamagnetic iron oxide nanoparticles or microRNA3686 can inhibit the expression of PLK1 and serve as therapeutic targets for PDAC [28, 29]. The overexpression of PLK1 is an adverse prognostic factor in various tumors and its expression is regulated by multiple factors. However, the interaction of circRNAs and PLK1 in PDAC
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remains to be investigated.
In this study, we found that hsa_circ_0000977 and PLK1 were aberrantly overexpressed in pancreatic cancer tissues.
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The aberrant expressions were closely associated with negative clinical characteristics and poor prognosis. Silencing hsa_circ_0000977 suppressed pancreatic cancer cell growth both in vitro and in vivo. We further demonstrated that hsa_circ_0000977/miR-874-3P/PLK1 axis plays an important role in regulating pancreatic cancer cell proliferation. Therefore, hsa_circ_0000977 overexpression may serve as a promising biomarker for early diagnosis and prognosis of PDAC and silencing hsa_circ_0000977 could be a therapeutic strategy.
Materials and methods Chemicals The DAB reagent kit was obtained from Boster Biological Technology Co. Ltd (Wuhan, China). Phosphate-buffered saline (PBS) was obtained from Wuhan Boster Biological Technology Co. Ltd. RIPA Lysis Buffer was obtained from Beyotime Institute of Biotechnology (Shanghai, China). Lipofectamine 2000 was obtained from Invitrogen (Camarillo, CA,
USA). The Cell Counting Kit-8 was obtained from Dojinodo (Shanghai, China). Enhanced BCA Protein Assay Kit was
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obtained from the Beyotime Institute of Biotechnology (Shanghai, China). Propidium iodide was obtained from Kaiji (Nanjing, China). Chemiluminescent HRP Substrate was obtained from Millipore (Billerica, MA, USA). TRIzol Reagent was obtained from Invitrogen. Prime Script RT reagent Kit, Mir-X miR First-Strand Synthesis Kit, and SYBR Premix Ex Taq II were obtained from TaKaRa (Dalian, China). The anti-PLK1, anti-cyclin D1, anti-bcl-2, anti-cleaved caspase-3
control vector were purchased from GeneChem (Shanghai, China). Cell culture
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monoclonal antibody (mAb) was obtained from Abcam (Shanghai, China). Lentiviral-hsa_circ_0000977-RNAi and the
Two pancreatic cell lines, AsPC-1 and PANC-1 (Chinese Academy of Sciences), were used in this study. The two cell lines were cultured in DMEM with 10% fetal bovine serum (FBS) and 1% antibiotics and maintained at 37°C in a
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humidified atmosphere with 5% CO2. Pancreatic cancer tissue specimens
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Fifty matched cancer and adjacent non-cancerous tissues, seventy-one cancer tissues, and five normal tissues were used in this study. Fifty matched cancer and adjacent non-cancerous tissues, fifty-one cancer tissues, and five normal tissues were obtained from the Institute of Hepatopancreatobiliary Surgery, Southwest Hospital, Third Military Medical University. The other twenty cancer tissues were obtained from the Department of Hepatobiliary Surgery, Zhujiang Hospital, Southern
specimens. Analyzing circRNA expression profile
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Medical University. The ethics committee of both ZhuJiang Hospital and Southwest Hospital approved the use of clinical
Twenty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues were analyzed using the
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circRNAs chips. The microarray hybridization and collection of data were performed by KangChen Bio-tech, Shanghai, China. The five most up- and down-regulated circRNAs and their hierarchical clustering analysis were performed based on
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their expression value using the Cluster and TreeView program. RNA extraction and qRT-PCR
Thirty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and eighteen cancer tissues were used to perform qRT-PCR. TRIzol Reagent (Invitrogen) was used to extract total RNA from these tissues. For circRNAs, RNase R was used to degrade linear RNA, which have poly (A), and amplified by divergent primer. qRT-PCR analysis on circular RNA and mRNA was performed using Prime Script RT reagent Kit (TaKaRa) and SYBR Premix Ex Taq II (TaKaRa). β-actin was used as an endogenous control. For miR-874-3P analysis, miRNA was treated with DNase I to eliminate genomic DNA and cDNA was synthesized by Mir-X miR First-Strand Synthesis Kit (TaKaRa). SYBR Premix Ex Taq II (TaKaRa) was used for qRT-PCR. The expression was normalized to RNU6-2. The 2-∆∆CT method was adopted to calculate relative expression of hsa_circ_0000977 and hsa-miR-874-3p. Cell transfection
AsPC-1 and PANC-1 cells were seeded in 6-well plates and cultured to 60-70% confluence before transfection.
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According to the manufacturer’s instructions, miRNA mimics, inhibitors (Sangon, China), and siRNA (GeneChem, China) were transiently transfected using Lipofectamine 2000 (Invitrogen) at a final concentration of 50 nM. The corresponding negative controls were transfected simultaneously under the same condition. Lentiviral-hsa_circ_0000977-RNAi and the negative control lentiviruses were purchased from GeneChem (Shanghai, China) and transfected according to the manufacturer’s protocol.
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Protein isolation and Western blot
Twelve matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and additional five normal tissues from above (qRT-PCR) were lysed using RIPA buffer according to manufacturer's instructions. 8% SDS-PAGE was used to separate protein extracts. After electrophoresis, protein extracts were transferred onto Nitrocellulose membranes
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(Biosharp) in 200 mA for 2 h, followed by blocking for another 2 h, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies for 2 h at room temperature. HRP activity was detected using Chemiluminescent
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HRP Substrate (Millipore, Billerica, MA, USA) and visualized with a UVP BioImaging system. The protein isolation and Western blot analysis on transfected pancreatic cells were performed following the same procedures. Cell proliferation assay
After transfection, cells were seeded in 96-well plates with 3000 cells per well. 10 µl of the Cell Counting Kit-8 reagent (Dojinodo, Shanghai, China) was added to each well and the OD value was measured after 4 h incubation. We
Cell cycle assay
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record the OD value of 24, 48, 72, and 96 h after transfection for statistical analysis.
For cell cycle analysis, transfected AsPC-1 and PANC-1 cells were fixed in 70% ethanol overnight at -20°C and
Colony formation assay
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stained with propidium iodide (Kaiji, Nanjing, China). Cell cycle assays were conducted at 48 h after transfection.
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One hundred transfected pancreatic cancer cells were seeded in 6-well plates. After 2 weeks of incubation, colonies (>200 cells per colony) were stained with Giemsa. The number of colonies was counted. Each experiment was performed in triplicate.
Luciferase reporter assay
HEK293T cells were seeded in 96-well plates and cultured to 50-70% confluence before transfection. The cells were divided into four groups: PLK1-3'UTR and hsa-miR-874-3p, PLK1-3'UTR and N.C, PLK1-3'UTR-muta and hsa-miR-874-3p, PLK1-3'UTR-muta and N.C. For every corresponding experimental group, 0.16 µg plasmids of PLK13’UTR and PLK13’UTR-muta, 5 pmol of hsa-miR-874-3p and N.C. were used. After 48 h incubation, Promega Dual-Luciferase system was used to detect firefly and Renilla luciferase activities. Using 100 µl Luciferase Assay Reagent II (LAR II) (Luciferase Assay Reagent, Progema) and subsequently 20 µl lysis buffer, firefly luciferase activities were measured as internal reference, and Renilla luciferase activities were also measured using 100 µl Stop & Glo® Reagent
(Luciferase Assay Reagent, Progema). Finally, the subtracted difference of firefly and Renilla luciferase activities were
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calculated as relative luciferase activity. Immunohistochemistry (IHC) Thirty matched pancreatic ductal adenocarcinoma and adjacent non-cancerous tissues, and additional fifty-three cancer tissues were used for paraffin embedding and sectioning. IHC analysis was performed under manufacturer’s instructions. Briefly, the slides were incubated with primary antibodies overnight at 4°C and then incubated with secondary antibodies at
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room temperature for 2 h. The expression was evaluated using a composite score obtained by multiplying the values of staining intensities (0, no staining; 1, weak staining; 2, moderate staining; 3, strong staining) and the percentage of positive cells (0, 0%; 1, <10%; 2, 10-50%; 3, > 50%). Fluorescence in situ hybridization (FISH)
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The hybridization was performed overnight with hsa_circ_0000977 and hsa-miR-874-3p probes. Specimens were analyzed on a Nikon inverted fluorescence microscope. The hsa_circ_0000977 probe for FISH was 5’-TGGAT GTTGT
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TTACT GATTC TGGCA ATTAA-3’ and the miR-874-3p probe for FISH was 5’-TCGGT CCCTC GGGCC AGGGC AG-3’. Xenografts experiments
Five-week-old female BALB/c nude mice were selected for this study. A total number of 2×106 AsPC-1 cells transfected with Lentiviral-hsa_circ_0000977-RNAi and negative control were subcutaneously injected into the back of nude mice. The
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volume and weight of tumors were measured after 20 days. The Institutional Animal Care and Use Committee of the Third Military Medical University Animal approved the experimental procedures. Statistical analysis
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Statistical analysis was accomplished using SPSS 22.0 (version 22.0, Chicago, IL, USA). The data was presented as means ± s.d. For quantitative data, Student’s t test and Mann-Whitney U test were used. For categorical data, Chi-square test was used. The
when P < 0.05.
RESULTS
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log-rank test was used to compare different survival curves by Kaplan-Meier analysis. Significant difference was considered
Informatics reveal circRNA hsa_circ_0000977/hsa-miR-874-3p/PLK1 axis Using high throughput microarray assay, we identified the expression profiles of dysregulated circRNAs. We found 289 circRNAs were aberrantly expressed with fold change ≥ 2.0 and P < 0.05. Among them, 128 circRNAs were up-regulated and 161 circRNAs were down-regulated. Fold change filtering displayed the differentially expressed circRNAs (Fig. 1A) and Volcano Plot Filtering identified differentially changed circRNAs with statistical difference between carcinoma and non-carcinoma tissues (Fig. 1B). Hierarchical clustering showed the five most up- and down-regulated
circRNAs between cancer and adjacent non-cancerous tissues (Fig. 1C). Then, we further confirmed the expression of these
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5 most up-regulated circRNAs in 5 matched PDAC and non-tumor specimens, and found that hsa_circ_0000977 was consistently and significantly increased in PDAC tissues as compared to matched controls by nearly 71 folds in the microarray assays (Supplementary Fig. 1). To investigate the potential miRNAs associated with hsa_circ_0000977, TargetScan and miRanda database were used, and the five most potentially complementary binding miRNAs were presented (Supplementary Table 1). MiRWalk 2.0, Gene ontology (GO) and KEGG enrichment analysis were used to investigate the
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potential target of miRNAs (Fig. 1D). Among these target miRNAs, we found that miR-874-3p has the potential to interact with PLK1. The microRNA response elements (MREs) are shown in Fig. 1E. These results suggested that hsa_circ_0000977 may have the potential to regulate the expression of PLK1 by sequestering miR-874-3p, in turn promoting the progression of pancreatic cancer.
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Overexpression of hsa_circ_0000977 is correlated with poor prognosis in PDAC patients
We examined the expression of hsa_circ_0000977 and miR-874-3p by qRT-PCR in 30 matched PDAC and adjacent
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non-cancer tissues. Gel electrophoresis and DNA sequence confirmed the PCR product of hsa_circ_0000977 (Fig. 2A). qRT-PCR showed higher expression of hsa-circ-0000977 and lower expression of miR-874-3p in cancer tissues than in matched non-tumor tissues (Fig. 2B, 2C). In pancreatic cancer tissues, hsa_circ_0000977 showed higher expression than miR-874-3p (Supplementary Fig.2). This was verified in the same batch of tissues, indicating that the expression of hsa-circ-0000977 and miR-874-3p had a negative correlation. Furthermore, the expression of hsa_circ_0000977 in 48
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pancreatic cancer tissues was examined. Based on the median value of expression, patients were classified into low and high expression groups. Compared to the low expression group, Kaplan-Meier survival curves showed that the patients with high expression of hsa_circ_0000977 had a low 5-year overall survival rate (12 VS 16.5 months, P=0.0403) (Fig. 2D). These
PDAC patients.
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results demonstrate that hsa_circ_0000977 is overexpressed in PDAC tissues and is associated with poor prognosis of
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hsa_circ_0000977 regulates cell proliferation and cell cycle by sequestering miR-874-3P in vitro The roles of hsa_circ_0000977 in PADC progression were further investigated. To block the hsa_circ_0000977 expression, two pancreatic cell lines, AsPC-1 and PANC-1, were transfected with hsa_circ_0000977 siRNA and the silencing efficiency was confirmed by qRT-PCR. After transfection, the expression of hsa_circ_0000977 and PLK1 decreased compared to the controls (Supplementary Fig. 3). Since PLK1 is involved in the initiation, maintenance, and completion of cell mitosis, and has interaction with hsa_circ_0000977, we assumed that hsa_circ_0000977 might also be involved in cell cycle regulation and cell proliferation. CCK-8 assay showed that hsa_circ_0000977 siRNA and miR-874-3P mimics induced a proliferation-suppressing effect, whereas the suppressing effect was reversed by an miR-874-3P inhibitor (Fig. 3A). Consistently, colony formation assays showed that hsa_circ_0000977 siRNA and miR-874-3P mimics decreased colony formation ability, and no significant difference was observed when hsa_circ_0000977 siRNA and miR-874-3P inhibitor were transfected simultaneously (Fig. 3B). Flow cytometry demonstrated that hsa_circ_0000977 siRNA and
miR-874-3P mimics induced G1/S arrest, and co-transfection of hsa_circ_0000977 siRNA and miR-874-3P inhibitor
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abrogated the cell cycle block effects (Fig. 3C). The above assays were also repeated in PANC-1 cell lines, which showed similar results (Supplementary Fig. 4). These results suggest that hsa_circ_0000977 modulates proliferation and the cell cycle as a miR-874-3P sponge in PDAC cells. hsa_circ_0000977 regulates PLK1 by sequestering miR-874-3p Bioinformatics analysis showed that both hsa_circ_0000977 and PLK1 contain mutual miRNA response elements (MREs)
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to miR-874-3p. We hypothesized that hsa_circ_0000977 exerts its biological function as a miR-874-3p sponge to regulate PLK1 expression. Dual-luciferase reporter assays were then performed to confirm whether there was a direct interaction between hsa_circ_0000977 and miR-874-3p, or between miR-874-3p and PLK1. The alignments of potential binding sites and their mutant types were constructed through bioinformatics analysis (Fig. 4A, 4B). There was significant reduction of
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firefly luciferase reporter activity for transfected hsa-miR-874-3p and hsa-circ-0000977 WT, and there was no notable change of luciferase reporter activity in the hsa-circ-0000977 mutant (Fig. 4C). Co-transfection of hsa-miR-874-3p and PLK1 WT also
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significantly decreased the firefly luciferase reporter activity (Fig. 4D). These results indicated that direct binding interactions existed between hsa_circ_0000977 and miR-874-3p, and between miR-874-3p and PLK1. qRT–PCR was used to investigate whether hsa-circ-0000977 regulates PLK1mRNA expression via hsa-miR-874-3p. Both hsa_circ_0000977 siRNA and miR-874-3p mimics significantly reduced the PLK1 mRNA expression in pancreatic cancer cells. There was no notable change in PLK1 expression when cells were simultaneously treated with an hsa_circ_0000977 siRNA and a
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miR-874-3p inhibitor, indicating that a miR-874-3p inhibitor could significantly rescue the decrease of PLK1 induced by silencing hsa_circ_0000977 (Fig. 4E, 4F). The interaction between hsa_circ_0000977 and miR-874-3p was further investigated by FISH analysis in AsPC-1 and PANC-1 cell lines. We found that hsa_circ_0000977 and miR-874-3p were
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co-localized in the cytoplasm, which suggested that hsa_circ_0000977 binds to miR-874-3p (Fig. 4G). Taken together, these results confirm our hypothesis that hsa_circ_0000977 regulates PLK1 through miR-874-3p in pancreatic cancer
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progression.
PLK1 is overexpressed in pancreatic cancer tissues and regulated by hsa_circ_0000977 Based on computational algorithms and clinical application significance, we chose PLK1 as the presumed target of hsa_circ_0000977/miR-874-3P. Twelve pairs of matched pancreatic cancer tissues and adjacent non-cancerous tissues, and 5 normal tissues were examined for the expression of PLK1. Through Western blot analysis, we found that PLK1 expression was significantly up-regulated in cancer tissues (Fig. 5A). To further investigate PLK1 expression in the tissues, we performed immunohistochemistry in 30 matched tissues. We found that PLK1 showed high expression in pancreatic cancer tissues in which hsa_circ_0000977 was also overexpressed (Fig. 5B). Furthermore, we found that PLK1 was up-regulated in 83 archival formalin-fixed, paraffin-embedded pancreatic cancer tissues, especially in advanced stage pancreatic cancer (Supplementary Fig. 5). The level of PLK1 expression was significantly associated with clinical stage, as patients with high PLK1 expression were more likely in advanced stage (Supplementary Table 2). Therefore, Western blot was performed to
examine whether hsa_circ_0000977 can affect PLK1 protein expression through miR-874-3P. In PDAC cell lines, both
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silencing of hsa_circ_0000977 or transfection with miR-874-3P mimic significantly reduced the expression of PLK1. PLK1 expression was rescued when miR-874-3p inhibitor was co-transfected with hsa_circ_0000977 siRNA (Fig. 5C). There were also signs that PLK1 could interact with other gene products, indicating that PLK1 may play an important role in other cancerous events such as apoptosis. The associated proteins were then examined. After knockdown of hsa_circ_0000977, cell cycle-related proteins cyclin D1 and bcl-2 were significantly decreased. However, apoptosis-related proteins cleaved
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caspase-3 were significantly increased (Fig. 5D). Finally, these results confirmed that overexpression of PLK1 was a significant feature of pancreatic cancer, and that hsa_circ_0000977 modulated PLK1 as a miR-874-3P sponge in PDAC. Knockdown of hsa_circ_0000977 inhibits pancreatic cell growth in vivo
Silencing hsa_circ_0000977 suppresses proliferation of pancreatic cancer cell in vitro. We investigated whether this is
with
low
hsa_circ_0000977
expression
via
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also true in vivo using a xenograft nude mouse model. AsPC-1 cells were used to establish a stable pancreatic cancer cell line Lentiviral-hsa_circ_0000977-RNAi.
The
biological
impact
of
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hsa_circ_0000977-RNAi was measured by CCK-8 assays and flow cytometry analysis. The results showed that Lentiviral-hsa_circ_0000977-RNAi significantly decreased hsa_circ_0000977 expressions, reduced cell proliferation and blocked cell cycle (Supplementary Fig. 6).
AsPC-1 cells with low or normal hsa_circ_0000977 expression induced by transfection with lentiviral or control vectors were subcutaneously implanted into nude mice. After 20 days, the weights of the tumors were measured. Tumors
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derived from low hsa_circ_0000977 expression group had lower weight than their negative controls (Fig. 6A). The expression level of PLK1 was examined by IHC. The results showed that, compared to negative controls, expression of PLK1 significantly decreased (Fig. 6B). These results demonstrate that hsa_circ_0000977 can suppress pancreatic cancer
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Discussion
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progression in vivo and decrease PLK1 expression.
CircRNAs are a novel class of extensive and stable endogenous RNAs that regulate gene expression in mammals [30-32]. The covalently closed loop structures render circRNAs more stable than liner RNA and insusceptible to RNA exonuclease or RNase [33]. CircRNAs mainly act as a miRNA sponge to exert their post-transcriptional functions as ceRNAs, which is more effective than the traditional anti-miRNA approach [8]. It has been elucidated that some circRNAs may be potential biomarkers for the diagnosis of gastric cancer, hepatocellular carcinoma, lung cancer, colon carcinoma, leukemia, and other cancers [34]. Because of the cell type or developmental stage specific characteristics, circRNAs may also play roles in PDAC progression and serve as valuable clinical biomarkers for PDAC. Compared to circRNAs, miRNAs have been well studied. Many studies have documented that dysregulation of miRNA is closely associated with tumorigenesis. miRNAs inhibit target protein translation by interacting with the untranslated region (3’-UTR) of target
mRNAs [35]. Previous studies have shown that miRNAs play diverse and significant roles in PDAC progression and
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metastasis [36]. In the ceRNA networks, circRNAs have strong inhibitory effect on miRNAs, and the interaction between circRNAs and miRNAs has already been observed to perform significant functions in a variety of cancers. In this study, hsa_circ_0000977 was found to be aberrantly up-regulated in pancreatic cancer tissues by high throughput microarray assay. This finding was further confirmed by qRT-PCR. Based on bioinformatics analysis, it was assumed that the hsa_circ_0000977/miR-874-3P/PLK1 axis plays a pivotal role in pancreatic cancer progression. Luciferase reporter system
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showed that miR-874-3P targets both hsa_circ_0000977 and PLK1, which provided direct evidence that hsa_circ_0000977 functions as a miR-874-3P sponge to modulate PLK1 expression. FISH analysis demonstrated that hsa_circ_0000977 and miR-874-3P primarily reside in the cytoplasm, and their co-location explained their post-transcriptional regulatory role. Cytological function experiment and flow cytometry revealed that hsa_circ_0000977 and miR-874-3P have reverse effects
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in cell phenotype and cell cycle. A miR-874-3P inhibitor could rescue biological changes induced by silencing of hsa_circ_0000977. Taken together, the study revealed that an hsa_circ_0000977/miR-874-3P/PLK1 axis exists in PDAC,
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and that hsa_circ_0000977 negatively regulates miR-874-3P. The aberrantly up-regulated hsa_circ_0000977 accompanied with by down-regulated miR-874-3P may be potentially used for early diagnosis and determining prognosis in pancreatic cancer patients.
PLK1 is overexpressed in various types of cancers, and dysregulation of PLK1 causes tumorigenesis by disrupting many events in cellular processes [37, 38]. Despite its important roles in the cell cycle, PLK1 also has mutual interactions
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with other pivotal molecules in cells [39]. Previous studies have shown that chemotherapy resistance, which is a common and early occurrence in pancreatic cancer, is often associated with PLK1 overexpression [40]. In this study, we confirmed that PLK1 was over-expressed in PDAC tissues and was related to histological grade and TNM stage. Hsa_circ_0000977
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promoted PDAC progression by up-regulating PLK1 expression through the hsa_circ_0000977/ miR-874-3P/PLK1 axis, and silencing of hsa_circ_0000977 inhibited PLK1 expression.
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In conclusion, this is the first study to investigate the regulatory function of hsa_circ_0000977 in PDAC and the interaction between hsa_circ_0000977, miRNA, and PLK1. Through the hsa_circ_0000977/miR-874-3P/PLK1 axis, hsa_circ_0000977 performed specific regulatory roles in the proliferation and progression of PDAC. Hsa_circ_0000977 could be a novel biomarker of poor prognosis in pancreatic cancer. In addition, the hsa_circ_0000977/miR-874-3P/PLK1 axis is a special signaling pathway, which could be a potential therapeutic target for pancreatic cancer patients.
Fundings This work was supported by the National Key R&D Program of China (No.2017YFC1308600); The National Key Research Program of China (2016YFC1201802); the National Nature Science Foundation of China (No.81672382, 81502550, 81371652); the Clinical Research Foundation of TMMU (No. SWH2015LC01, SWH2016JCZD01).
Acknowledgement： ：We thank Dr. Dong-Hua Yang(Fox Chase Cancer Center, Philadelphia) for helping to revise the
manuscript.
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Conflict of interests： ：The authors declare that they have no competing interests.
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[37] U. Holtrich, G. Wolf, A. Brauninger, T. Karn, B. Bohme, H. Rubsamen-Waigmann, K. Strebhardt, Induction and down-regulation of PLK, a human serine/threonine kinase expressed in proliferating cells and tumors, Proceedings of the National Academy of Sciences of the United States of America 91 (1994) 1736-1740. [38] V. Thalhammer, L.A. Lopez-Garcia, D. Herrero-Martin, R. Hecker, D. Laubscher, M.E. Gierisch, M. Wachtel, P. Bode, P. Nanni, B. Blank, E. Koscielniak, B.W. Schafer, PLK1 phosphorylates PAX3-FOXO1, the inhibition of which triggers regression of alveolar Rhabdomyosarcoma, Cancer research 75 (2015) 98-110. [39] M.A. Amin, G. Itoh, K. Iemura, M. Ikeda, K. Tanaka, CLIP-170 recruits PLK1 to kinetochores during early mitosis for chromosome alignment, Journal of cell science 127 (2014) 2818-2824. [40] A. Jimeno, B. Rubio-Viqueira, N.V. Rajeshkumar, A. Chan, A. Solomon, M. Hidalgo, A fine-needle aspirate-based vulnerability assay identifies polo-like kinase 1 as a mediator of gemcitabine resistance in pancreatic cancer, Molecular
cancer therapeutics 9 (2010) 311-318.
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Fig legends Fig. 1. The identification of circRNA expression profiles and prediction of hsa_circ_0000977 functional mechanism (A)Scatter plot of circRNAs expression in PDAC and adjacent non-carcinoma tissues. The circRNAs above the top green line and below the bottom green line are those up or down-regulated more than 2.0 folds. (B) Volcano plot visualizes the different
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expression in these two groups. The red dots represent more than 2.0 fold changes (log2 scaled) of circRNAs and with statistical significance (P <0.05). (C) Hierarchical cluster analysis of the top five up- and down-regulated circRNAs in 20 pairs of matched tissues. (D) The association of circRNA-miRNA-mRNAs in the five most up-regulated circRNAs. In this network, the interaction of the five most up-regulated circRNAs (green circle), 12 targeted miRNAs (orange circle) and 207
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microRNA response elements (MREs) of hsa-miR-874-3p.
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targeted mRNAs (green triangle) were exhibited. (E) TargetScan and miRanda database confirm hsa_circ_0000977 contains
Fig. 2. The expression of hsa_circ_0000977 and miR-874-3p
(A) PCR product of hsa_circ_0000977 in 1.5% agarose gelelectrophoresis and DNA sequencing analysis. DL2000 DNA marker was used (from up to down, 2000,1000, 750, 500, 250, 100 bp). (B) qRT–PCR on Hsa_circ_0000977 expression in 30 paired pancreatic cancer tissues and adjacent non-cancer tissues. (C) qRT–PCR on miR-874-3p expression in 30 paired pancreatic cancer tissues and
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adjacent non-cancer tissues. (D) The Kaplan–Meier survival analysis and log-rank test show up-regulation of hsa_circ_0000977 correlates with low 5-year overall survival rates. The median survival time for patients with high and low expression of
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hsa_circ_0000977 was 12 months as compared to 16.5 months respectively (P = 0.0403).
Fig. 3. Silencing hsa_circ_0000977 suppresses pancreatic cancer cell proliferation and induces cell cycle arrest in AsPC-1 cell
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(A) CCK-8 assay. hsa_circ_0000977 siRNA and miR-874-3P mimics suppresses cancer cell proliferation, miR-874-3P inhibitor abolishes the suppression effect of has_circ-0000977 knockdown. OD values are obtained at 24,48,72,96 hours after transtection. (B) Colony formation assay. hsa_circ_0000977 siRNA and miR-874-3P mimics reduced cancer cell colony forming ability, miR-874-3P inhibitor restores the inhibiting effect of has_circ-0000977 siRNA. (C) Flow cytometric analysis. Cell block were observed when hsa_circ_0000977 siNRA and miR-874-3P mimics were transfected. Co-transfection of has_circ-0000977 siRNA and miR-874-3P inhibitor have no obvious impact on cell cycle. Experiments were repeated 3 times and data were presented as means±s.d. NC, negative control. *P<0.05, **P<0.01, ***P<0.001.
Fig.4. The interaction of hsa_circ_0000977, miR-874-3p and PLK1 (A) miRNA response elements (MREs) is showed by which hsa_circ_0000977 sequesters miR-874-3p. Mutations were
generated in MREs. (B) Putative miR-874-3p binding sites in the 3′-UTR of PLK1 mRNA is showed. The sequence in the
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PLK1 3′-UTR at the complementary sites of miR-874-3p were mutated. (C, D) Dual luciferase reporter show significant reduction of luciferase activity of the wild-type and luciferase activity is restored by the mutant sequence. Compared to mutant group, the luciferase activity of hsa_circ_0000977 wild type has decreased 23% and the luciferase activity of PLK1 wild type has decreased 30%. The relative luciferase activities were normalized with Renilla activity. (E, F) qRT-PCR illustrates inhibition of circ-0000977 and overexpression of miR-874-3p degrades PLK1 mRNA, and miR-874-3p inhibitor
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restores PLK1 mRNA expression. (G) The co-localization of hsa_circ_0000977 and miR-874-3p in pancreatic cancer cells by FISH. *P<0.05, **P<0.01, ***P<0.001.
Fig. 5. PLK1 overexpressed in pancreatic cancer tissues and hsa_circ_0000977 regulates PLK1 expression by acting
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(A)Western blot analysis in 12 pairs of pancreatic cancer tissues show PLK1 is up-regulated in cancer tissues compared to
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adjacent noncancerous and normal tissues. Aj, adjacent non-cancerous tissue; T, pancreatic cancer; N, normal tissues. (B) Immunohistochemistry on PLK1 expression in pancreatic cancer and adjacent non-cancerous tissues. (C)Silencing of hsa_circ_0000977 by siRNA inhibiting PLK1expression，overexpression of miR874-3p by mimics suppress PLK1 expression and miR874-3p inhibitor restore the PLK1expression which is suppressed by hsa_circ_0000977 siRNA. (D)
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silencing of hsa_circ_0000977 increase caspase-3 expression and decrease cyclin D1and bcl-2 expression.
Fig. 6. Knockdown of hsa_circ_0000977 suppresses pancreatic cancer growth and PLK1 expression (A)Representative graph show tumor growth and the mean tumor weights 30 days after subcutaneously implanted AsPC-1
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cells. (B) Compared negative control, The PLK1 expression was decreased.
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ACCEPTED MANUSCRIPT Highlights: 1. Aberrantly up-regulated hsa_circ_0000977 play an important role in PDAC proliferation.
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2. hsa_circ_0000977 and miR-874-3P exert their post transcriptional regulation roles in PDAC.
3. hsa_circ_0000977 act as miR-874-3P sponge to exert its oncogenic roles.
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4. hsa_circ_0000977/ miR-874-3P/PLK1 axis is established and confirmed in PDAC
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5. Knockdown of hsa_circ_0000977 surely suppress PDAC growth by inhibiting
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PLK1 in vivo.