- Email: [email protected]
MDM2 via miR-342-3p
Journal Pre-proof Dysbindin promotes pancreatic ductal adenocarcinoma metastasis by activating NFκB/MDM2 via miR-342-3p Donglie Zhu, Shi Zheng, Cheng Fang, Xin Guo, Dandan Han, Mingyao Tang, Hang Fu, Mingzuo Jiang, Ning Xie, Yongzhan Nie, Xuebiao Yao, Yong Chen PII:
S0304-3835(20)30102-6
DOI:
https://doi.org/10.1016/j.canlet.2020.02.033
Reference:
CAN 114715
To appear in:
Cancer Letters
Received Date: 10 October 2019 Revised Date:
11 February 2020
Accepted Date: 26 February 2020
Please cite this article as: D. Zhu, S. Zheng, C. Fang, X. Guo, D. Han, M. Tang, H. Fu, M. Jiang, N. Xie, Y. Nie, X. Yao, Y. Chen, Dysbindin promotes pancreatic ductal adenocarcinoma metastasis by activating NF-κB/MDM2 via miR-342-3p, Cancer Letters, https://doi.org/10.1016/j.canlet.2020.02.033. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2,
metastasis
Revision completed date: 2020/2/8
Dysbindin promotes pancreatic ductal adenocarcinoma metastasis by activating NF-κB/MDM2 via miR-342-3p Donglie Zhu1#, Shi Zheng1#, Cheng Fang2#, Xin Guo3, Dandan Han1, Mingyao Tang1, Hang Fu1, Mingzuo Jiang4, Ning Xie5, Yongzhan Nie4*, Xuebiao Yao6* and Yong Chen1* 1
Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an,
Shaanxi, 710032, China; 2
Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 200438,
China; 3
Department of Endoscopic Surgery, 986th Military Hospital, Fourth Military Medical University,
Xi’an, Shaanxi, 710054, China; 4
State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive
Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, Shaanxi, 710032, China; 5
Department of Gastroenterology, the Second Affiliated Hospital of Xi’an Jiaotong University,
Xi’an, Shaanxi, 710004, China; 6
Department of Hefei Laboratory for Physical Sciences at Microscale, School of Life Science,
University of Science and Technology of China, Hefei, China.
#
These authors contributed equally to this work.
* Correspondence to: Yong Chen, E-mail: [email protected] or Xuebiao Yao, E-mail: [email protected] or Yongzhan Nie, E-mail: [email protected]
1
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2,
metastasis
2
1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies and remains the fourth leading cause of cancer-related death worldwide[1, 2]. The five-year survival rate of PDAC patients is less than 5%, and the average patient survival is only 6-9 months after diagnosis. PDAC is predicted to become the second leading cause of cancer-related death in the future[3]. Moreover, approximately 80–85% of affected patients are not candidates for a radical resection due to distant metastasis or locally advanced disease[4, 5]. The protein dysbindin has diverse physiological roles, particularly in the nervous system. DTNBP1, the gene that encodes dysbindin, is a susceptibility gene related to schizophrenia[6], and dysbindin protein levels are lower in the hippocampus of schizophrenia patients compared with the healthy people[7]. However, the role of dysbindin in solid tumours is not yet clear. MDM2 is the primary negative regulator of p53 and plays a key role in regulating p53 transcriptional activity[8, 9]. MDM2 is overexpressed in various cancers and may play a key role in epithelial-mesenchymal transition (EMT), an essential step in the progression to metastatic disease[10, 11]. The NF-κB family of ubiquitously expressed dimeric sequence-specific transcription factors is involved in regulating cellular growth, controlling apoptosis, and stimulating invasion/metastasis[12, 13]. The NF-κB signalling pathway is regulated by IκB and its kinase, IκB kinase (IKK)[14-16]. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level. MiRNAs play a crucial role in diverse biological processes, such as cell growth, proliferation, development and apoptosis[17, 18]. MiR-342-3p is downregulated in human cancers, such as hepatocellular carcinoma (HCC)[19] and colon cancer[20]. However, the relationship between dysbindin and miR-342-3p in PDAC has not been reported. Our previous study found that dysbindin levels were higher in PDAC patients than in control individuals and that this protein regulated cell growth via the PI3K pathway[21, 22]. However, we recently determined that dysbindin may be closely involved in PDAC metastasis and invasion. However, the mechanisms by which dysbindin upregulation and activation are involved in PDAC have not been elucidated. Thus, in this study, we investigated the signalling process leading to 3
dysbindin upregulation and elucidated the underlying mechanism through which dysbindin promotes PDAC metastasis and invasion.
2. Methods and materials 2.1 Cell culture and tissue samples The human PDAC cell lines Aspc-1 and Bxpc-3 were purchased from iCell Bioscience Inc. (Shanghai). Human pancreatic ductal epithelial cells (HPDE6c-7) and other PDAC cell lines were a gift from Dr Qu. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 10% foetal bovine serum (FBS, Biological Industries) at 37°C in 5% CO2. PDAC and paired adjacent normal tissues were collected from 20 patients (Xi Jing Hospital, Xi’an, China) from February 2018 to January 2019. The Ethics Committee of Xi Jing Hospital approved this study.
2.2 Tissue microarray construction and immunohistochemistry A tissue microarray containing 63 PDAC specimens and 57 adjacent noncancerous tissues was used in this study. The percentage of positive cells was scored as 0 (< 10%), 1 (10–40%), 2 (40–60%) or 3 (> 60%), and the intensity of the immunostaining was scored as 0 (no staining), 1 (weak), 2 (moderate) or 3 (strong). The product of the above two scores was used as the final immunohistochemistry (IHC) staining score. A score of 1–6 represented low expression, and 7–9 was considered high expression. IHC staining was performed on formalin-fixed, paraffin-embedded tissues. The tissue sections were heated for 1 h at 60°C, dewaxed and rehydrated. Then, antigen retrieval was performed, and the sections were blocked with 3% H2O2. Subsequently, 10% goat serum was used to block nonspecific staining. Appropriate primary and secondary antibodies were used, and DAB substrate was used for the chromogenic reaction.
2.3 Cell transfection A miR-342-3p mimic and a negative control mimic were purchased from RiboBio 4
(Guangzhou, China). siRNAs targeting dysbindin and MDM2 were synthesized by GenePharma (Shanghai, China). And the interference sequences are provided in the supplementary files. Transfections were performed with Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen).
2.4 Lentivirus HEK293T cells were used for virus packaging with a mixture of pHelper 1.0 vector (packaging plasmid) and pHelper 2.0 vector (envelope plasmid) (GeneChem, Shanghai, China) and Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 h, the virus-containing supernatants were collected. The shRNA sequences are provided in the supplementary files. The GV493 vector (hU6-MCS-CBh-gcGFP-IRES-puromycin, GeneChem, Shanghai,
China)
was
used
to
generate
dysbindin-shRNA.
The
GV341
vector
(Ubi-MCS-3FLAG-SV40-puromycin, GeneChem, Shanghai, China) was used to generate stably transfected cells overexpressing dysbindin. An empty vector was used as a negative control. Stable cell lines were generated by selection with 2 µg/ml puromycin for 15 days.
2.5 RNA extraction and qRT-PCR analysis Total RNA was extracted from cells and tissues using TRIzol Reagent (Invitrogen), and 1 µg of RNA was reverse transcribed to generate cDNA using a PrimeScript™ RT Master Mix kit (Takara, Japan). mRNA expression levels were detected by qPCR using a SYBR Premix Ex Taq reagent kit (Takara, Japan) following the manufacturer’s instructions. The primer sequences are provided in the supplementary files. The PCR conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Bio-Rad CFX96 software was used to analyse the PCR results.
2.6 Western blot analysis and regents Total protein lysates were harvested from PDAC cells using RIPA buffer supplemented with proteinase and phosphatase inhibitors. Total protein was quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Total protein (30 µg) was separated by 10-12% SDS-PAGE 5
and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were blocked in 5% nonfat milk for 1-2 h at room temperature and then incubated overnight with the following primary antibodies: dysbindin antibody (Abcam, monoclonal, 1:1000), MDM2 antibody (CST, monoclonal, 1:1000), IKKβ antibody (CST, monoclonal, 1:1000), phospho-IKKβ antibody (CST, monoclonal, 1:1000), IκBα antibody (CST, monoclonal, 1:1000), phospho-IκBα antibody (CST, monoclonal, 1:1000), NF-κB p65 antibody (CST, monoclonal, 1:1000), phospho-NF-κB p65 antibody (CST, monoclonal, 1:1000), and GAPDH antibody (CST, monoclonal, 1:2000). On the next day, the membranes were incubated with secondary antibodies for 1 h. All experiments were conducted in triplicate. PDAC cells were treated with curcumin (20 µM; Selleck Chemicals, USA) and increasing concentrations of TNF-α (10 or 20 ng/ml; Sigma-Aldrich).
2.7 Dual luciferase reporter gene assay The 3′UTR of the human dysbindin gene was amplified using PCR and cloned into the pGL3 vector to generate pGL3-dysbindin-wild type (WT). The mutated (Mut) dysbindin 3′UTR was constructed using site-directed mutagenesis to generate pGL3-dysbindin-Mut. Because we identified three putative miR-342-3p binding sites in the 3’UTR of dysbindin mRNA, we mutated all three putative binding sites at the same time and constructed two reporter plasmids: pGL3-dysbindin-WT and pGL3-dysbindin-Mut. HEK293T cells were grown to 70-80% confluence in a six-well plate in DMEM (HyClone) supplemented with 10% FBS (Biological Industries) at 37°C in 5% CO2. Then, the cells were cotransfected with either pGL3-dysbindin-WT or pGL3-dysbindin-Mut and miR-342-3p mimic or negative control for 48 h. The Dual Luciferase Reporter Assay System (Promega USA) was used to assess luciferase activity after 48 h of transfection according to the manufacturer's instructions.
2.8 Cell migration and invasion assays For cell migration assays, 5×104 cells were seeded in serum-free medium in the upper chamber (Millipore, USA), and medium containing 20% FBS was placed in the bottom chamber. After 24-48 h, the cells on the upper side of the membrane were removed carefully with a cotton swab, and those in the lower side of the membrane were fixed with paraformaldehyde and 6
stained with a crystal violet solution. To investigate cell invasion, Matrigel (Sigma)-coated transwell chambers (Millipore, USA) were used, and 5×104 cells were seeded in serum-free medium in the upper chamber. After 48-72 h of incubation, the cells were treated as previously described.
2.9 Animal experiments A tail vein injection assay was performed to evaluate the in vivo metastatic ability of dysbindin. A total of thirty-two male nude mice were divided into four groups of 8 rats each: the Aspc-1-vector, Aspc-1-dysbindin, Panc-1-vector and Panc-1-shDysbindin groups. All nude mice were housed in a specific pathogen-free (SPF) laboratory. Eight weeks after model creation, the nude mice were killed. The mouse lungs were harvested, fixed with formalin and subjected to haematoxylin and eosin (H&E) staining to assess the development of metastatic nodules. All mice were maintained under SPF conditions. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures performed in studies involving animals were in accordance with the ethical standards of The Fourth Military Medical University.
2.10 Statistical analysis Statistical analysis was performed using SPSS 22.0. Student’s t test was used to analyse differences between two groups. Pearson’s correlation coefficient was used to analyse the correlations involving expression. The Kaplan-Meier method and log-rank test were used to analyse the overall survival rate. Correlations between MDM2 expression and clinicopathological characteristics were evaluated using the chi-square test. The Cox proportional hazards model was used for univariate and multivariate analyses. P<0.05 was considered to indicate statistical significance.
3. Results 3.1 Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. First, we detected dysbindin expression in PDAC cells and human pancreatic ductal 7
epithelial cells. The data showed that dysbindin expression was higher in PDAC cells than in human pancreatic ductal epithelial cells (figure 1A). Among the PDAC cells, dysbindin expression was relatively higher in Panc-1 and Capan-2 cells (figure 1A). To evaluate whether dysbindin promotes PDAC metastasis and invasion in vitro and in vivo, we used a lentivirus to overexpress or silence dysbindin. Then, we used a transwell assay to evaluate cell motility. Dysbindin protein and mRNA expression levels are shown in figures 1B and 1C. Transwell assays showed that upregulating dysbindin promoted the metastasis and invasion of Aspc-1 and Bxpc-3 cells (figure 1D). Conversely, knocking down dysbindin expression decreased the migration and invasion of Panc-1 and Capan-2 cells (figure 1E). Transduced cells were injected into the tail vein of nude mice to observe the in vivo effects of dysbindin on PDAC metastasis. Stable cell lines with dysbindin overexpression or knockdown were established. As shown in figures 1G and 1H, the number of metastatic lung nodules and the incidence of lung metastasis were increased when dysbindin was upregulated, while these effects were completely reversed when dysbindin was downregulated. These studies indicate that dysbindin promotes PDAC metastasis and invasion in vitro and in vivo.
3.2 Dysbindin overexpression increases MDM2 expression in PDAC, and MDM2 knockdown decreases PDAC metastasis and attenuates dysbindin-enhanced metastasis and invasion. To further elucidate the mechanism by which dysbindin promotes PDAC metastasis and invasion, a whole-transcript human gene expression array was used to comprehensively identify differentially expressed mRNAs in Panc-1 cells with and without dysbindin dysregulation, with a particular focus on genes involved in cancer. Dysbindin downregulation substantially reduced the expression of numerous genes, and we selected one of the most downregulated metastasis-related genes, MDM2, for further analysis (figure 2A). MDM2 is a primary inhibitor of p53, promotes tumour metastasis and is associated with invasive ductal breast carcinoma and ovarian cancer[23, 24]. Considering the important role of MDM2 in tumour metastasis, we wondered whether MDM2 participates in dysbindin-mediated PDAC metastasis. As shown in figure 2B, dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) had 8
higher MDM2 expression than control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). Moreover, MDM2 expression was lower in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA)
than
in
control
cells
(Panc-1-control-siRNA
and
Capan-2-control-siRNA) (figure 2C). We silenced MDM2 expression using siRNA, and the resulting MDM2 protein and mRNA expression levels are shown in figure 2D. MDM2 knockdown significantly decreased the metastasis and invasion of Capan-2 and Bxpc-3 cells (figure 2E). Dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) were further transfected with control-siRNA or MDM2-siRNA (figure 2F). We found that MDM2 knockdown significantly attenuated dysbindin-enhanced metastasis and invasion (figure 2G, H). These results suggest that dysbindin overexpression increases MDM2 expression and that MDM2 knockdown decreases PDAC metastasis and invasion and attenuates dysbindin-enhanced metastasis and invasion.
3.3
MDM2
expression
is
upregulated
in
PDAC
tissues
and
correlates
with
clinicopathological characteristics; dysbindin expression levels are positively correlated with MDM2 expression in PDAC tissues. To further determine the association between MDM2 expression and PDAC, we first detected MDM2 expression in PDAC and paired adjacent noncancerous tissues and found that it was higher in PDAC tissues than in paired adjacent noncancerous tissues (figure 3A, B). Moreover, PDAC tissue microarrays were used to visualise MDM2 expression in PDAC and paired adjacent noncancerous tissues; the results showed higher MDM2 expression in PDAC tissues than in paired adjacent noncancerous tissues (figure 3C-E). Kaplan-Meier survival curves revealed that MDM2 overexpression was associated with shorter overall survival in PDAC patients (P < 0.05) (figure 3F). As shown in Table 1, MDM2 expression was significantly correlated with PDAC tumour size and histological grade (differentiation). Cox regression analyses indicated that MDM2 overexpression and perineural invasion were independent and significant predictors of prognosis in PDAC patients (Table 2). These results reveal correlations of MDM2 expression with PDAC tumour size and histological grade (differentiation) and the overall survival of PDAC patients. Next, we further ascertained the association between 9
dysbindin and MDM2 expression in human PDAC tissues. IHC results showed that dysbindin and MDM2 expression were both increased in PDAC tissues but decreased in paired adjacent noncancerous tissues (figure 3H). Pearson’s correlation analysis revealed a positive correlation between dysbindin and MDM2 expression in human PDAC tissues (r=0.639, p<0.01) (figure 3G).
3.4 Dysbindin activates the NF-κB signalling pathway in PDAC cells, and the dysbindin-induced overexpression of MDM2 is dependent on the NF-κB signalling pathway. To further study how dysbindin increases MDM2 expression in PDAC, we carried out Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the potential functions of dysbindin. As shown in figure 4A, KEGG pathway analysis identified 20 pathways enriched in the mRNAs dysregulated by dysbindin. Of these, one of the most enriched pathways was the NF-κB signalling pathway, which is known to be involved in regulating cancer metastasis, so we focused on this signalling pathway. To validate the results of the KEGG pathway enrichment analyses, we examined the protein levels of NF-κB signalling pathway-related
genes
in
dysbindin-overexpressing
cells
(Bxpc-3-LV-dysbindin
cells),
dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA cells) and control cells. The results showed that p-IKKβ, p-IκBα and p-p65 expression levels were significantly increased in dysbindin-overexpressing cells but decreased in dysbindin-silenced cells, while IKKβ, IκBα and p65 expression did not change significantly (figure 4B-D). These results indicate that dysbindin promotes activation of the NF-κB signalling pathway in PDAC cells. To investigate whether the NF-κB signalling pathway is involved in dysbindin-induced MDM2 upregulation, we first examined the relationship between this pathway and MDM2. As shown in figure 4E, MDM2 protein levels decreased in Panc-1 and Aspc-1 cells compared to control cells after treatment with curcumin, an inhibitor of the NF-κB signalling pathway. Conversely, MDM2 protein levels were higher in Panc-1 and Capan-2 cells than in control cells after treatment with increasing concentrations of TNF-α, a known activator of the NF-κB signalling pathway (figure 4F). To determine whether NF-κB signalling pathway activation is required for dysbindin-induced 10
MDM2 overexpression, we blocked the NF-κB signalling pathway with curcumin in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin cells) and control cells. We found that MDM2 protein expression levels did not change considerably in dysbindin-overexpressing cells compared with control cells (figure 4G). These findings indicate that the NF-κB signalling pathway can regulate MDM2 expression and that dysbindin-induced MDM2 overexpression is dependent on the NF-κB signalling pathway.
3.5 Dysbindin, a direct target of miR-342-3p, is negatively regulated by miR-342-3p in PDAC. Because dysbindin plays an important role in PDAC development, we wanted to determine how dysbindin is regulated in PDAC. MiRNAs have been reported to be closely involved in PDAC, but there is no published research on the relationship between miRNAs and dysbindin. Therefore, we aimed to elucidate whether miRNAs are involved in the regulation of dysbindin. We predicted candidate miRNAs targeting dysbindin using TargetScan software. As shown in figure 5A, we screened the top 5 miRNAs that potentially regulate dysbindin according to the context++ score generated by TargetScan software. Of these, two miRNAs had conserved target sites in the 3’UTR of dysbindin, and three had poorly conserved target sites. The predicted putative miRNA binding sites in the 3’UTR of dysbindin are shown in figure 5B. Based on these data, we transfected Aspc-1 cells with five candidate miRNA mimics (miR-199a-5p, miR-199b-5p, miR-182-5p, miR-377-3p and miR-342-3p). As shown in figure 5C, Western blotting showed that miR-342-3p had the most significant inhibitory effect on dysbindin. We used different cells (Aspc-1, Capan-2 and Bxpc-3) to validate that dysbindin protein levels were lower in PDAC cells transfected with miR-342-3p mimic than in control cells (figure 5D). Taken together, the data supported the selection of miR-342-3p for further experiments. Luciferase assays were performed to validate the direct binding of miR-342-3p to the 3′UTR of dysbindin. As shown in figure 5E, we identified three putative miR-342-3p binding sites in the 3’UTR of dysbindin mRNA. Luciferase assays showed that miR-342-3p overexpression significantly suppressed the WT dysbindin 3’UTR reporter construct but not the mutant construct. These data suggest that dysbindin is negatively regulated by miR-342-3p in PDAC and a direct target of miR-342-3p. 11
3.6 MiR-342-3p is downregulated in PDAC tissues, and dysbindin is significantly negatively correlated with miR-342-3p. The Cancer Genome Atlas (TCGA) data showed that in PDAC, miR-342-3p is downregulated (figure 6A), and dysbindin is upregulated (figure 6B). To validate miR-342-3p and dysbindin expression in PDAC, we measured miR-342-3p expression and dysbindin mRNA levels in 8 paired PDAC and adjacent noncancerous tissues by qRT-PCR. As shown in figure 6C, miR-342-3p expression was higher in adjacent noncancerous tissues than in PDAC tissues. Conversely, dysbindin expression was higher in PDAC tissues than in adjacent noncancerous tissues. Subsequently, we measured miR-342-3p expression in three PDAC cell lines (Panc-1, Bxpc-3 and Aspc-1) and a normal pancreatic ductal epithelial cell line (HPDE6c-7). As shown in figure 6D, miR-342-3p was downregulated in PDAC cell lines and upregulated in HPDE6c-7 cells. Dysbindin expression was significantly negatively correlated with miR-342-3p (figure 6E). In addition, TCGA data showed that low miR-342-3p expression levels were correlated with poor overall survival among PDAC patients (figure 6F). These data indicate that miR-342-3p is downregulated in PDAC and that dysbindin is significantly negatively correlated with miR-342-3p.
3.7 The antitumour effect of miR-342-3p on PDAC cell lines is decreased by dysbindin overexpression, and miR-342-3p decreases MDM2 expression. To determine the effect of miR-342-3p on PDAC metastasis and invasion, transwell assays were conducted with Aspc-1, Capan-2 and Panc-1 cells transfected with mimic control or miR-342-3p mimic. As shown in figure 7A, the miR-342-3p-transfected PDAC cells (Aspc-1, Capan-2 and Panc-1) were less metastatic and invasive than the control cells. We confirmed that miR-342-3p decreased dysbindin expression, and we next wondered whether dysbindin overexpression can reverse the antitumour effect of miR-342-3p in PDAC cell lines. Therefore, a dysbindin overexpression vector was constructed and transfected into Aspc-1 cells, and dysbindin expression levels in different groups are shown in figure 7B. Transwell assays showed that dysbindin overexpression significantly restored the migration and invasion capacities of 12
miR-342-3p-transfected cell lines (figure 7C). As the terminal molecule in the signalling axis, MDM2 plays a crucial role in NF-κB/MDM2 signalling, but it was unclear whether miR-342-3p affects MDM2 expression. Therefore, we detected MDM2 protein expression levels in PDAC cells (Panc-1 and Bxpc-3) transfected with mimic control or miR-342-3p mimic and found that MDM2 expression was lower in the miR-342-3p-transfected PDAC cell lines (Panc-1 and Bxpc-3) than in the control cells (figure 7D). In summary, these results show that the antitumour effect of miR-342-3p in PDAC cell lines is diminished by dysbindin overexpression and that miR-342-3p can decrease MDM2 expression.
4. Discussion Over the years, considerable efforts have been made to explore the molecular mechanism of PDAC invasion and metastasis; nonetheless, the mechanisms that lead to PDAC metastasis remain unknown. Our previous study reported that dysbindin expression is correlated with tumour size, tumour differentiation and clinical stage, indicating that dysbindin might be closely involved in PDAC metastasis. In this study, we found that dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. However, the precise mechanism by which dysbindin promotes PDAC metastasis remains unknown. To elucidate the potential molecular mechanism by which dysbindin promotes PDAC metastasis, we analysed differentially expressed cancer-related genes using a whole-transcript human gene expression array and selected MDM2 as a candidate gene. MDM2 is involved in the pathogenesis of different types of cancer, such as breast and cervical cancer[25, 26]. MDM2 is also related to tumour invasiveness in endometrial cancer[27]. In this study, we found that dysbindin overexpression increases MDM2 expression and that MDM2 knockdown decreases PDAC metastasis and invasion and attenuates the effects of dysbindin on these processes. These results suggest that dysbindin might promote PDAC cell invasion by upregulating MDM2. Therefore, we explored the potential signalling pathways involved in the regulation of MDM2 expression by dysbindin and found evidence for the potential participation of the NF-κB signalling pathway. We found that dysbindin upregulation increased key components of the NF-κB signalling pathway, indicating that dysbindin can activate this pathway. Dysbindin was reported to 13
promote NF-κB transcriptional activity in the nucleus in neuroblastoma cells[28], which is consistent with our results. When we blocked the NF-κB signalling pathway using curcumin, MDM2 expression was downregulated. In addition, we activated the NF-κB signalling pathway by upregulating TNF-α and MDM2 expression. These data show that dysbindin-induced MDM2 overexpression might depend on the NF-κB signalling pathway. Dysbindin plays a crucial role in PDAC metastasis. It is necessary to determine the underlying mechanism of dysbindin upregulation. A number of miRNAs are downregulated in PDAC and thus might regulate PDAC-related genes by affecting important signalling pathways[29]. In this study, miR-342-3p was identified as a candidate that potentially targets dysbindin. Recent studies have shown that miR-342-3p is downregulated in non-small cell lung cancer, nasopharyngeal carcinoma and cervical cancer[30-32]. We found that miR-342-3p expression negatively correlated with dysbindin expression and that dysbindin is a direct target of miR-342-3p. Rescue experiments demonstrated that the antitumour effect of miR-342-3p was decreased after dysbindin overexpression, indicating that dysbindin is a crucial target of miR-342-3p in PDAC. In conclusion, we demonstrated that dysbindin is regulated by miR-342-3p in PDAC and promotes PDAC metastasis and invasion by activating the NF-κB/MDM2 signalling axis. Thus, our findings clarify the mechanism of dysbindin upregulation in PDAC and how dysbindin promotes PDAC metastasis; ultimately, these data provide a therapeutic target for clinical intervention.
5. Acknowledgements This study was supported by grants from the National Key R&D Program of China (No. 2017YFC1308600), Key Research and Development Program of Shaanxi Province (No. 2017ZDXM-SF-024), and National Natural Science Foundation of China (No. 81670563). The authors are grateful for technical support from Mingzuo Jiang (State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University) and Ning Xie (Department of Gastroenterology, the Second Affiliated Hospital of Xi’an Jiaotong University).
14
6. Conflict of interest The authors have declared that no conflicts of interest exist.
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of GABA Neuron and Ion Permeation Transcripts in the Developing Hippocampus, Frontiers in genetics, 8 (2017) 28. [8] S. Wang, Y. Zhao, A. Aguilar, D. Bernard, C.Y. Yang, Targeting the MDM2-p53 Protein-Protein Interaction for New Cancer Therapy: Progress and Challenges, Cold Spring Harbor perspectives in medicine, 7 (2017) a026245. [9] J.D. Oliner, A.Y. Saiki, S. Caenepeel, The Role of MDM2 Amplification and Overexpression in Tumorigenesis, Cold Spring Harbor perspectives in medicine, 6 (2016) a026336. [10] C.M. Eischen, Role of Mdm2 and Mdmx in DNA repair, Journal of molecular cell biology, 9 (2017) 69-73. [11] O. Karni-Schmidt, M. Lokshin, C. Prives, The Roles of MDM2 and MDMX in Cancer, Annual review of pathology, 11 (2016) 617-644. [12] C.H. Lee, Y.T. Jeon, S.H. Kim, Y.S. Song, NF-kappaB as a potential molecular target for cancer therapy, BioFactors (Oxford, England), 29 (2007) 19-35. [13] H.J. Kim, N. Hawke, A.S. Baldwin, NF-kappaB and IKK as therapeutic targets in cancer, Cell death and differentiation, 13 (2006) 738-747. [14] S. Wang, Z. Liu, L. Wang, X. Zhang, NF-kappaB signaling pathway, inflammation and colorectal cancer, Cellular & molecular immunology, 6 (2009) 327-334. [15] A. Oeckinghaus, S. Ghosh, The NF-kappaB family of transcription factors and its regulation, Cold Spring Harbor perspectives in biology, 1 (2009) a000034. [16] V. Tergaonkar, M. Pando, O. Vafa, G. Wahl, I. Verma, p53 stabilization is decreased upon NFkappaB activation: a role for NFkappaB in acquisition of resistance to chemotherapy, Cancer cell, 1 (2002) 493-503. 16
[17] M. Falasca, M. Kim, I. Casari, Pancreatic cancer: Current research and future directions, Biochimica et biophysica acta, 1865 (2016) 123-132. [18] F.B. Abreu, X. Liu, G.J. Tsongalis, miRNA analysis in pancreatic cancer: the Dartmouth experience, Clinical chemistry and laboratory medicine, 55 (2017) 755-762. [19] Y. Gao, S.G. Zhang, Z.H. Wang, J.C. Liao, Down-regulation of miR-342-3p in hepatocellular carcinoma tissues and its prognostic significance, European review for medical and pharmacological sciences, 21 (2017) 2098-2102. [20] K. Tao, J. Yang, Z. Guo, Y. Hu, H. Sheng, H. Gao, H. Yu, Prognostic value of miR-221-3p, miR-342-3p and miR-491-5p expression in colon cancer, American journal of translational research, 6 (2014) 391-401. [21] X. Guo, X. Lv, C. Fang, X. Lv, F. Wang, D. Wang, J. Zhao, Y. Ma, Y. Xue, Q. Bai, X. Yao, Y. Chen, Dysbindin as a novel biomarker for pancreatic ductal adenocarcinoma identified by proteomic profiling, Int J Cancer, 139 (2016) 1821-1829. [22] C. Fang, X. Guo, X. Lv, R. Yin, X. Lv, F. Wang, J. Zhao, Q. Bai, X. Yao, Y. Chen, Dysbindin promotes progression of pancreatic ductal adenocarcinoma via direct activation of PI3K, Journal of molecular cell biology, 9 (2017) 504-515. [23] X. Chen, J. Qiu, D. Yang, J. Lu, C. Yan, X. Zha, Y. Yin, MDM2 promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9, PloS one, 8 (2013) e78794. [24] Y. Chen, D.D. Wang, Y.P. Wu, D. Su, T.Y. Zhou, R.H. Gai, Y.Y. Fu, L. Zheng, Q.J. He, H. Zhu, B. Yang, MDM2 promotes epithelial-mesenchymal transition and metastasis of ovarian cancer SKOV3 cells, British journal of cancer, 117 (2017) 1192-1201. 17
[25] X. Lu, C. Yan, Y. Huang, D. Shi, Z. Fu, J. Qiu, Y. Yin, Mouse double minute 2 (MDM2) upregulates Snail expression and induces epithelial-to-mesenchymal transition in breast cancer cells in vitro and in vivo, Oncotarget, 7 (2016) 37177-37191. [26] A. Roszak, M. Misztal, A. Sowinska, P.P. Jagodzinski, Murine Double-Minute 2 Homolog Single Nucleotide Polymorphisms 285 and 309 in Cervical Carcinogenesis, Molecular diagnosis & therapy, 19 (2015) 235-244. [27] L. Liu, L. Yang, H. Chang, Y.N. Chen, F. Zhang, S. Feng, J. Peng, C.C. Ren, X.A. Zhang, CP31398 attenuates endometrial cancer cell invasion, metastasis and resistance to apoptosis by downregulating MDM2 expression, International journal of oncology, 54 (2019) 942-954. [28] C. Fu, D. Chen, R. Chen, Q. Hu, G. Wang, The Schizophrenia-Related Protein Dysbindin-1A Is Degraded and Facilitates NF-Kappa B Activity in the Nucleus, PloS one, 10 (2015) e0132639. [29] C. Vorvis, M. Koutsioumpa, D. Iliopoulos, Developments in miRNA gene signaling pathways in pancreatic cancer, Future oncology (London, England), 12 (2016) 1135-1150. [30] X. Xue, X. Fei, W. Hou, Y. Zhang, L. Liu, R. Hu, miR-342-3p suppresses cell proliferation and migration by targeting AGR2 in non-small cell lung cancer, Cancer letters, 412 (2018) 170-178. [31] Z. Cui, Y. Zhao, microRNA-342-3p targets FOXQ1 to suppress the aggressive phenotype of nasopharyngeal carcinoma cells, BMC cancer, 19 (2019) 104. [32] X.R. Li, H.J. Chu, T. Lv, L. Wang, S.F. Kong, S.Z. Dai, miR-342-3p suppresses proliferation, migration and invasion by targeting FOXM1 in human cervical cancer, FEBS letters, 588 (2014) 3298-3307. 18
Figure legends
Figure 1. Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. (A) Western blot analysis showing dysbindin expression in PDAC cells and human pancreatic ductal epithelial cells. The data are presented as the mean ± SEM. (B) Western blotting and qRT-PCR analysis showing dysbindin expression in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). The data are presented as the mean ± SEM. (C) After Panc-1 and Capan-2 cells were transfected with LV-shDysbindin (sh1 and sh2) or LV-shVector, Western blotting and qRT-PCR analysis were performed to measure dysbindin expression. The data are presented as the mean ± SEM. (D) Overexpression of dysbindin enhanced Aspc-1 and Bxpc-3 cell migration and invasion. The data are presented as the mean ± SEM. (E) Dysbindin knockdown decreased Panc-1 and Capan-2 cell migration and invasion. The data are presented as the mean ± SEM. (F) In vivo metastasis assays. Four stable cell lines (Aspc-1-LV-vector, Aspc-1-LV-dysbindin, Panc-1-LV-shvector and Panc-1-sh2dysbindin) were injected into the tail vein of nude mice. Representative H&E staining of the lungs from the above four groups is shown. Scale bars: top, 200 µm; bottom, 50 µm. (G, H) The incidence of lung metastasis and the number of metastatic lung nodules in nude mice from the above four groups after 8 weeks are shown. *P<0.05, **P<0.01, ***P<0.001.
Figure 2. Dysbindin overexpression increases MDM2 expression in PDAC, and MDM2 knockdown significantly attenuates dysbindin-enhanced metastasis and invasion. (A) A heatmap 19
was generated from the whole-transcript human gene expression array analysis of total RNA extracted from the Panc-1-dysbindin-siRNA and control groups. Red represents relatively lower expression, and green indicates relatively higher expression. (B) Western blot analysis showing MDM2 and dysbindin protein levels in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). The data are presented as the mean ± SEM. (C) Western blot analysis showing MDM2 and dysbindin protein levels in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA) and control cells (Panc-1-control-siRNA and Capan-2-control-siRNA). The data are presented as the mean ± SEM. (D) After Capan-2 and Bxpc-3 cells were transfected with MDM2-siRNA or control-siRNA, Western blotting and qRT-PCR analysis were performed to determine MDM2 expression. The data are presented as the mean ± SEM. (E) MDM2 knockdown decreased Capan-2 and Bxpc-3 cell migration and invasion. The data are presented as the mean ± SEM. (F) After dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) were transfected with MDM2-siRNA or control-siRNA, Western blot analysis was performed to determine MDM2 and dysbindin protein levels. (G, H) MDM2 knockdown attenuated dysbindin-enhanced metastasis and invasion. The data are presented as the mean ± SEM. **P<0.01, ***P<0.001.
Figure 3. MDM2 expression is upregulated in PDAC tissues and correlates with clinicopathological characteristics, and dysbindin expression is positively correlated with MDM2 expression in PDAC tissues. (A) Western blot analysis showing MDM2 protein levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (B) qRT-PCR analysis showing MDM2 mRNA levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (C) MDM2 expression was detected on a tissue microarray containing 63 PDAC specimens and 57 adjacent noncancerous tissues. Representative IHC staining profiles of MDM2 in different groups are shown. ANT, adjacent noncancerous tissue; T, tumour tissue. Scale bars: top, 200 µm; bottom, 50 µm. (D) MDM2 expression levels in 63 PDAC tissues and 57 adjacent noncancerous tissues were analysed by Pearson's chi-square test. (E) Analysis of the IHC scores for MDM2 in PDAC 20
tissues and adjacent noncancerous tissues. ANT, adjacent noncancerous tissue; T, tumour tissue. The data are presented as the mean ± SEM. (F) Kaplan-Meier survival analysis showing the overall survival of PDAC patients with high or low MDM2 expression (n=63). (G) The association between dysbindin and MDM2 IHC scores in 20 PDAC tissues was analysed by Pearson’s correlation analysis (r=0.639, p<0.01). (H) Representative IHC staining profiles of dysbindin and MDM2 in PDAC tissues (n=20) and adjacent noncancerous tissues. Scale bars: top, 100 µm; bottom, 50 µm. ***P<0.001.
Figure 4. In PDAC cells, dysbindin activates the NF-κB signalling pathway, which regulates MDM2. Dysbindin-induced MDM2 overexpression is dependent on the NF-κB signalling pathway. (A) The top 20 significantly enriched biological processes identified by KEGG pathway analysis. The NF-κB signalling pathway was one of the most enriched pathways. (B-D) Western blot analysis showing the protein levels of NF-κB signalling pathway-related genes (IKKβ, p-IKKβ, IκBα, p-IκBα, p65 and p-p65) in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA), dysbindin-overexpressing cells (Bxpc-3-LV-dysbindin) and control cells. The data are presented as the mean ± SEM. (E) After PDAC cells (Panc-1 and Aspc-1) were treated with the NF-κB inhibitor curcumin (20 µM) for 24 h, Western blot analysis was performed to determine MDM2 and p-p65 protein levels in the curcumin-treated and DMSO (control)-treated groups. The data are presented as the mean ± SEM. (F) After PDAC cells (Panc-1 and Capan-2) were treated with control or increasing concentrations (10 or 20 ng/ml) of TNF-α, an NF-κB signalling pathway activator, Western blot analysis was performed to determine MDM2 and p-p65 protein levels in each group. The data are presented as the mean ± SEM. (G) Western blot analysis showing MDM2 and p-p65 protein levels in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) treated with curcumin (20 µM) for 24 h. The data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 5. Dysbindin, a direct target of miR-342-3p, is negatively regulated by miR-342-3p in PDAC. (A) The top 5 predicted candidate miRNAs (miR-199a-5p, miR-199b-5p, miR-182-5p, 21
miR-377-3p and miR-342-3p) targeting dysbindin according to TargetScan software. (B) Predicted pairing to the target region in the 3’UTR of dysbindin (top) and the five predicted candidate miRNAs (bottom). (C) Western blot analysis showing dysbindin protein levels in Aspc-1 cells transfected with the five candidate miRNA mimics (miR-199a-5p, miR-199b-5p, miR-182-5p, miR-377-3p and miR-342-3p). The data are presented as the mean ± SEM. (D) After PDAC cells (Aspc-1, Capan-2 and Bxpc-3) were transfected with miR-342-3p mimic or mimic control, Western blot analysis was performed to determine dysbindin protein levels. The data are presented as the mean ± SEM. (E) Three predicted binding sites of miR-342-3p in the 3’UTR of dysbindin mRNA. MiR-342-3p decreased the luciferase activity of the WT dysbindin reporter but not the mutant reporter. The data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 6. MiR-342-3p is downregulated in PDAC, and dysbindin is significantly negatively correlated with miR-342-3p. (A) MiR-342-3p is downregulated in PDAC according to The Cancer Genome Atlas (TCGA) data. NP, normal pancreas tissue; PC, pancreatic cancer tissue. (B) Dysbindin expression is upregulated in PDAC according to TCGA data. N, noncancerous tissue; T, tumour tissue. (C) qRT-PCR analysis showing miR-342-3p and dysbindin mRNA levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (D) qRT-PCR analysis showing miR-342-3p expression levels in PDAC cell lines (Panc-1, Bxpc-3 and Aspc-1) and a normal pancreatic ductal epithelial cell line (HPDE6c-7). The data are presented as the mean ± SEM. (E) The association between dysbindin mRNA and miR-342-3p expression was analysed by Pearson’s correlation coefficient (r=-0.6685, p<0.01). (F) Kaplan-Meier survival analysis showing the overall survival of PDAC patients with high or low miR-342-3p expression from TCGA data. *P<0.05, **P<0.01, ***P<0.001.
Figure 7. The antitumour effect of miR-342-3p on PDAC cell lines is diminished by dysbindin overexpression, and miR-342-3p decreases MDM2 expression. (A) MiR-342-3p-transfected PDAC cells (Aspc-1, Capan-2 and Panc-1) were less metastatic and invasive than control cells. The data are presented as the mean ± SEM. (B) Western blot analysis showing dysbindin protein 22
levels in the Aspc-1-vector, Aspc-1-miR-342-mimic and Aspc-1-miR-342-mimic+dysbindin groups. The data are presented as the mean ± SEM. (C) Transwell assays showing that dysbindin overexpression reverses the ability of miR-342-3p to inhibit PDAC cell (Aspc-1) migration and invasion. The data are presented as the mean ± SEM. (D) Western blot analysis showing MDM2 expression in miR-342-3p-transfected PDAC cells (Panc-1 and Bxpc-3) and control cells. The data are presented as the mean ± SEM. (E) Proposed model by which the NF-κB/MDM2 signalling axis is activated by dysbindin, which is regulated by miR-342-3p in PDAC cells. *P<0.05, **P<0.01, ***P<0.001.
23
Table1. Correlations between MDM2 expression and clinical characteristics in patients with PDAC. Variable
Patient number (n=63)
MDM2 expression high
low
(n=34)
(n=29)
P-value (χ2 test)
Age (years) ≤60
24
15
9
＞60
39
19
20
Male
36
24
12
Female
27
10
17
Head
37
20
17
Body/tail
26
14
12
≤4
42
21
21
＞4
21
13
8
N0
35
16
19
N1
25
15
10
Unknown
3
0.191
Gender
0.06
Tumor location
0.506
Tumor size (cm)
0.000* .000*
Nodal status
Perineural invasion
0.423
Absent
36
20
16
Present
27
14
13
Ⅰ,Ⅱ
34
15
19
Ⅲ
29
19
10
0.609
Histological Grade (differentiation)
*Statistically significant. PP-value < 0.05 are in bold.
0.000* .000*
Table2. Univariate and multivariate analyses of prognostic parameters for survival in PDAC patients. Variable
Univariate analysis HR
95% CI
P-value
MDM2 expression (high vs. low)
2.085
1.175-3.699
0.012*
Age (≤ (≤60 years vs. >60 years) years)
1.278
0.721- 2.268
0.401
Gender (male vs. female) female)
0.878
0.5-1.541
0.651
Tumor location (head vs. body/tail)
1.229
0.7-2.157
0.473
Tumor size (≤4 cm vs. >4 >4 cm)
1.589
0.873-2.895
0.13
Nodal status(N0 status(N0 vs. N1)
1.479
0.841-2.6
0.174
Perineural invasion(absent invasion(absent vs. present)
0.434
0.242-0.78
0.005*
Histological Grade (differentiation)
1.425
0.816-2.489
0.213
Variable
Multivariate analysis HR
95% CI
P-value
MDM2 expression (high vs. low)
2.523
1.276-4.987
0.008*
Perineural invasion(absent invasion(absent vs. present)
0.337
0.167-0.681
0.002*
Univariate analysis and multivariate analyses were analyzed by Cox proportional hazards model. HR, hazard ratio; 95% CI, 95% confidence interval. *Statistically significant. P-value < 0.05 are in bold.
1. Dysbindin could promote PDAC metastasis and invasion in vitro and in vivo. 2. Dysbindin could promote metastasis of PDAC via NF-κB/MDM2 signal axis. 3. MDM2 is upregulated in PDAC tissues and correlated with clinicopathological characteristics of PDAC. 4. Dysbindin is a direct target of miR-342-3p. 5. Dysbindin overexpression could decrease the antitumor effect of miR-342-3p.
The authors have declared that no conflict interests exist.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2, metastasis
S0304-3835(20)30102-6
DOI:
https://doi.org/10.1016/j.canlet.2020.02.033
Reference:
CAN 114715
To appear in:
Cancer Letters
Received Date: 10 October 2019 Revised Date:
11 February 2020
Accepted Date: 26 February 2020
Please cite this article as: D. Zhu, S. Zheng, C. Fang, X. Guo, D. Han, M. Tang, H. Fu, M. Jiang, N. Xie, Y. Nie, X. Yao, Y. Chen, Dysbindin promotes pancreatic ductal adenocarcinoma metastasis by activating NF-κB/MDM2 via miR-342-3p, Cancer Letters, https://doi.org/10.1016/j.canlet.2020.02.033. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2020 Published by Elsevier B.V.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2,
metastasis
Revision completed date: 2020/2/8
Dysbindin promotes pancreatic ductal adenocarcinoma metastasis by activating NF-κB/MDM2 via miR-342-3p Donglie Zhu1#, Shi Zheng1#, Cheng Fang2#, Xin Guo3, Dandan Han1, Mingyao Tang1, Hang Fu1, Mingzuo Jiang4, Ning Xie5, Yongzhan Nie4*, Xuebiao Yao6* and Yong Chen1* 1
Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an,
Shaanxi, 710032, China; 2
Eastern Hepatobiliary Surgery Hospital, Second Military Medical University, Shanghai, 200438,
China; 3
Department of Endoscopic Surgery, 986th Military Hospital, Fourth Military Medical University,
Xi’an, Shaanxi, 710054, China; 4
State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive
Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University, Xi’an, Shaanxi, 710032, China; 5
Department of Gastroenterology, the Second Affiliated Hospital of Xi’an Jiaotong University,
Xi’an, Shaanxi, 710004, China; 6
Department of Hefei Laboratory for Physical Sciences at Microscale, School of Life Science,
University of Science and Technology of China, Hefei, China.
#
These authors contributed equally to this work.
* Correspondence to: Yong Chen, E-mail: [email protected] or Xuebiao Yao, E-mail: [email protected] or Yongzhan Nie, E-mail: [email protected]
1
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2,
metastasis
2
1. Introduction Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive solid malignancies and remains the fourth leading cause of cancer-related death worldwide[1, 2]. The five-year survival rate of PDAC patients is less than 5%, and the average patient survival is only 6-9 months after diagnosis. PDAC is predicted to become the second leading cause of cancer-related death in the future[3]. Moreover, approximately 80–85% of affected patients are not candidates for a radical resection due to distant metastasis or locally advanced disease[4, 5]. The protein dysbindin has diverse physiological roles, particularly in the nervous system. DTNBP1, the gene that encodes dysbindin, is a susceptibility gene related to schizophrenia[6], and dysbindin protein levels are lower in the hippocampus of schizophrenia patients compared with the healthy people[7]. However, the role of dysbindin in solid tumours is not yet clear. MDM2 is the primary negative regulator of p53 and plays a key role in regulating p53 transcriptional activity[8, 9]. MDM2 is overexpressed in various cancers and may play a key role in epithelial-mesenchymal transition (EMT), an essential step in the progression to metastatic disease[10, 11]. The NF-κB family of ubiquitously expressed dimeric sequence-specific transcription factors is involved in regulating cellular growth, controlling apoptosis, and stimulating invasion/metastasis[12, 13]. The NF-κB signalling pathway is regulated by IκB and its kinase, IκB kinase (IKK)[14-16]. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression at the posttranscriptional level. MiRNAs play a crucial role in diverse biological processes, such as cell growth, proliferation, development and apoptosis[17, 18]. MiR-342-3p is downregulated in human cancers, such as hepatocellular carcinoma (HCC)[19] and colon cancer[20]. However, the relationship between dysbindin and miR-342-3p in PDAC has not been reported. Our previous study found that dysbindin levels were higher in PDAC patients than in control individuals and that this protein regulated cell growth via the PI3K pathway[21, 22]. However, we recently determined that dysbindin may be closely involved in PDAC metastasis and invasion. However, the mechanisms by which dysbindin upregulation and activation are involved in PDAC have not been elucidated. Thus, in this study, we investigated the signalling process leading to 3
dysbindin upregulation and elucidated the underlying mechanism through which dysbindin promotes PDAC metastasis and invasion.
2. Methods and materials 2.1 Cell culture and tissue samples The human PDAC cell lines Aspc-1 and Bxpc-3 were purchased from iCell Bioscience Inc. (Shanghai). Human pancreatic ductal epithelial cells (HPDE6c-7) and other PDAC cell lines were a gift from Dr Qu. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, HyClone) supplemented with 10% foetal bovine serum (FBS, Biological Industries) at 37°C in 5% CO2. PDAC and paired adjacent normal tissues were collected from 20 patients (Xi Jing Hospital, Xi’an, China) from February 2018 to January 2019. The Ethics Committee of Xi Jing Hospital approved this study.
2.2 Tissue microarray construction and immunohistochemistry A tissue microarray containing 63 PDAC specimens and 57 adjacent noncancerous tissues was used in this study. The percentage of positive cells was scored as 0 (< 10%), 1 (10–40%), 2 (40–60%) or 3 (> 60%), and the intensity of the immunostaining was scored as 0 (no staining), 1 (weak), 2 (moderate) or 3 (strong). The product of the above two scores was used as the final immunohistochemistry (IHC) staining score. A score of 1–6 represented low expression, and 7–9 was considered high expression. IHC staining was performed on formalin-fixed, paraffin-embedded tissues. The tissue sections were heated for 1 h at 60°C, dewaxed and rehydrated. Then, antigen retrieval was performed, and the sections were blocked with 3% H2O2. Subsequently, 10% goat serum was used to block nonspecific staining. Appropriate primary and secondary antibodies were used, and DAB substrate was used for the chromogenic reaction.
2.3 Cell transfection A miR-342-3p mimic and a negative control mimic were purchased from RiboBio 4
(Guangzhou, China). siRNAs targeting dysbindin and MDM2 were synthesized by GenePharma (Shanghai, China). And the interference sequences are provided in the supplementary files. Transfections were performed with Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen).
2.4 Lentivirus HEK293T cells were used for virus packaging with a mixture of pHelper 1.0 vector (packaging plasmid) and pHelper 2.0 vector (envelope plasmid) (GeneChem, Shanghai, China) and Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. After 48 h, the virus-containing supernatants were collected. The shRNA sequences are provided in the supplementary files. The GV493 vector (hU6-MCS-CBh-gcGFP-IRES-puromycin, GeneChem, Shanghai,
China)
was
used
to
generate
dysbindin-shRNA.
The
GV341
vector
(Ubi-MCS-3FLAG-SV40-puromycin, GeneChem, Shanghai, China) was used to generate stably transfected cells overexpressing dysbindin. An empty vector was used as a negative control. Stable cell lines were generated by selection with 2 µg/ml puromycin for 15 days.
2.5 RNA extraction and qRT-PCR analysis Total RNA was extracted from cells and tissues using TRIzol Reagent (Invitrogen), and 1 µg of RNA was reverse transcribed to generate cDNA using a PrimeScript™ RT Master Mix kit (Takara, Japan). mRNA expression levels were detected by qPCR using a SYBR Premix Ex Taq reagent kit (Takara, Japan) following the manufacturer’s instructions. The primer sequences are provided in the supplementary files. The PCR conditions were as follows: 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Bio-Rad CFX96 software was used to analyse the PCR results.
2.6 Western blot analysis and regents Total protein lysates were harvested from PDAC cells using RIPA buffer supplemented with proteinase and phosphatase inhibitors. Total protein was quantified using a Pierce BCA Protein Assay Kit (Thermo Scientific, USA). Total protein (30 µg) was separated by 10-12% SDS-PAGE 5
and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were blocked in 5% nonfat milk for 1-2 h at room temperature and then incubated overnight with the following primary antibodies: dysbindin antibody (Abcam, monoclonal, 1:1000), MDM2 antibody (CST, monoclonal, 1:1000), IKKβ antibody (CST, monoclonal, 1:1000), phospho-IKKβ antibody (CST, monoclonal, 1:1000), IκBα antibody (CST, monoclonal, 1:1000), phospho-IκBα antibody (CST, monoclonal, 1:1000), NF-κB p65 antibody (CST, monoclonal, 1:1000), phospho-NF-κB p65 antibody (CST, monoclonal, 1:1000), and GAPDH antibody (CST, monoclonal, 1:2000). On the next day, the membranes were incubated with secondary antibodies for 1 h. All experiments were conducted in triplicate. PDAC cells were treated with curcumin (20 µM; Selleck Chemicals, USA) and increasing concentrations of TNF-α (10 or 20 ng/ml; Sigma-Aldrich).
2.7 Dual luciferase reporter gene assay The 3′UTR of the human dysbindin gene was amplified using PCR and cloned into the pGL3 vector to generate pGL3-dysbindin-wild type (WT). The mutated (Mut) dysbindin 3′UTR was constructed using site-directed mutagenesis to generate pGL3-dysbindin-Mut. Because we identified three putative miR-342-3p binding sites in the 3’UTR of dysbindin mRNA, we mutated all three putative binding sites at the same time and constructed two reporter plasmids: pGL3-dysbindin-WT and pGL3-dysbindin-Mut. HEK293T cells were grown to 70-80% confluence in a six-well plate in DMEM (HyClone) supplemented with 10% FBS (Biological Industries) at 37°C in 5% CO2. Then, the cells were cotransfected with either pGL3-dysbindin-WT or pGL3-dysbindin-Mut and miR-342-3p mimic or negative control for 48 h. The Dual Luciferase Reporter Assay System (Promega USA) was used to assess luciferase activity after 48 h of transfection according to the manufacturer's instructions.
2.8 Cell migration and invasion assays For cell migration assays, 5×104 cells were seeded in serum-free medium in the upper chamber (Millipore, USA), and medium containing 20% FBS was placed in the bottom chamber. After 24-48 h, the cells on the upper side of the membrane were removed carefully with a cotton swab, and those in the lower side of the membrane were fixed with paraformaldehyde and 6
stained with a crystal violet solution. To investigate cell invasion, Matrigel (Sigma)-coated transwell chambers (Millipore, USA) were used, and 5×104 cells were seeded in serum-free medium in the upper chamber. After 48-72 h of incubation, the cells were treated as previously described.
2.9 Animal experiments A tail vein injection assay was performed to evaluate the in vivo metastatic ability of dysbindin. A total of thirty-two male nude mice were divided into four groups of 8 rats each: the Aspc-1-vector, Aspc-1-dysbindin, Panc-1-vector and Panc-1-shDysbindin groups. All nude mice were housed in a specific pathogen-free (SPF) laboratory. Eight weeks after model creation, the nude mice were killed. The mouse lungs were harvested, fixed with formalin and subjected to haematoxylin and eosin (H&E) staining to assess the development of metastatic nodules. All mice were maintained under SPF conditions. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All procedures performed in studies involving animals were in accordance with the ethical standards of The Fourth Military Medical University.
2.10 Statistical analysis Statistical analysis was performed using SPSS 22.0. Student’s t test was used to analyse differences between two groups. Pearson’s correlation coefficient was used to analyse the correlations involving expression. The Kaplan-Meier method and log-rank test were used to analyse the overall survival rate. Correlations between MDM2 expression and clinicopathological characteristics were evaluated using the chi-square test. The Cox proportional hazards model was used for univariate and multivariate analyses. P<0.05 was considered to indicate statistical significance.
3. Results 3.1 Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. First, we detected dysbindin expression in PDAC cells and human pancreatic ductal 7
epithelial cells. The data showed that dysbindin expression was higher in PDAC cells than in human pancreatic ductal epithelial cells (figure 1A). Among the PDAC cells, dysbindin expression was relatively higher in Panc-1 and Capan-2 cells (figure 1A). To evaluate whether dysbindin promotes PDAC metastasis and invasion in vitro and in vivo, we used a lentivirus to overexpress or silence dysbindin. Then, we used a transwell assay to evaluate cell motility. Dysbindin protein and mRNA expression levels are shown in figures 1B and 1C. Transwell assays showed that upregulating dysbindin promoted the metastasis and invasion of Aspc-1 and Bxpc-3 cells (figure 1D). Conversely, knocking down dysbindin expression decreased the migration and invasion of Panc-1 and Capan-2 cells (figure 1E). Transduced cells were injected into the tail vein of nude mice to observe the in vivo effects of dysbindin on PDAC metastasis. Stable cell lines with dysbindin overexpression or knockdown were established. As shown in figures 1G and 1H, the number of metastatic lung nodules and the incidence of lung metastasis were increased when dysbindin was upregulated, while these effects were completely reversed when dysbindin was downregulated. These studies indicate that dysbindin promotes PDAC metastasis and invasion in vitro and in vivo.
3.2 Dysbindin overexpression increases MDM2 expression in PDAC, and MDM2 knockdown decreases PDAC metastasis and attenuates dysbindin-enhanced metastasis and invasion. To further elucidate the mechanism by which dysbindin promotes PDAC metastasis and invasion, a whole-transcript human gene expression array was used to comprehensively identify differentially expressed mRNAs in Panc-1 cells with and without dysbindin dysregulation, with a particular focus on genes involved in cancer. Dysbindin downregulation substantially reduced the expression of numerous genes, and we selected one of the most downregulated metastasis-related genes, MDM2, for further analysis (figure 2A). MDM2 is a primary inhibitor of p53, promotes tumour metastasis and is associated with invasive ductal breast carcinoma and ovarian cancer[23, 24]. Considering the important role of MDM2 in tumour metastasis, we wondered whether MDM2 participates in dysbindin-mediated PDAC metastasis. As shown in figure 2B, dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) had 8
higher MDM2 expression than control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). Moreover, MDM2 expression was lower in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA)
than
in
control
cells
(Panc-1-control-siRNA
and
Capan-2-control-siRNA) (figure 2C). We silenced MDM2 expression using siRNA, and the resulting MDM2 protein and mRNA expression levels are shown in figure 2D. MDM2 knockdown significantly decreased the metastasis and invasion of Capan-2 and Bxpc-3 cells (figure 2E). Dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) were further transfected with control-siRNA or MDM2-siRNA (figure 2F). We found that MDM2 knockdown significantly attenuated dysbindin-enhanced metastasis and invasion (figure 2G, H). These results suggest that dysbindin overexpression increases MDM2 expression and that MDM2 knockdown decreases PDAC metastasis and invasion and attenuates dysbindin-enhanced metastasis and invasion.
3.3
MDM2
expression
is
upregulated
in
PDAC
tissues
and
correlates
with
clinicopathological characteristics; dysbindin expression levels are positively correlated with MDM2 expression in PDAC tissues. To further determine the association between MDM2 expression and PDAC, we first detected MDM2 expression in PDAC and paired adjacent noncancerous tissues and found that it was higher in PDAC tissues than in paired adjacent noncancerous tissues (figure 3A, B). Moreover, PDAC tissue microarrays were used to visualise MDM2 expression in PDAC and paired adjacent noncancerous tissues; the results showed higher MDM2 expression in PDAC tissues than in paired adjacent noncancerous tissues (figure 3C-E). Kaplan-Meier survival curves revealed that MDM2 overexpression was associated with shorter overall survival in PDAC patients (P < 0.05) (figure 3F). As shown in Table 1, MDM2 expression was significantly correlated with PDAC tumour size and histological grade (differentiation). Cox regression analyses indicated that MDM2 overexpression and perineural invasion were independent and significant predictors of prognosis in PDAC patients (Table 2). These results reveal correlations of MDM2 expression with PDAC tumour size and histological grade (differentiation) and the overall survival of PDAC patients. Next, we further ascertained the association between 9
dysbindin and MDM2 expression in human PDAC tissues. IHC results showed that dysbindin and MDM2 expression were both increased in PDAC tissues but decreased in paired adjacent noncancerous tissues (figure 3H). Pearson’s correlation analysis revealed a positive correlation between dysbindin and MDM2 expression in human PDAC tissues (r=0.639, p<0.01) (figure 3G).
3.4 Dysbindin activates the NF-κB signalling pathway in PDAC cells, and the dysbindin-induced overexpression of MDM2 is dependent on the NF-κB signalling pathway. To further study how dysbindin increases MDM2 expression in PDAC, we carried out Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis to determine the potential functions of dysbindin. As shown in figure 4A, KEGG pathway analysis identified 20 pathways enriched in the mRNAs dysregulated by dysbindin. Of these, one of the most enriched pathways was the NF-κB signalling pathway, which is known to be involved in regulating cancer metastasis, so we focused on this signalling pathway. To validate the results of the KEGG pathway enrichment analyses, we examined the protein levels of NF-κB signalling pathway-related
genes
in
dysbindin-overexpressing
cells
(Bxpc-3-LV-dysbindin
cells),
dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA cells) and control cells. The results showed that p-IKKβ, p-IκBα and p-p65 expression levels were significantly increased in dysbindin-overexpressing cells but decreased in dysbindin-silenced cells, while IKKβ, IκBα and p65 expression did not change significantly (figure 4B-D). These results indicate that dysbindin promotes activation of the NF-κB signalling pathway in PDAC cells. To investigate whether the NF-κB signalling pathway is involved in dysbindin-induced MDM2 upregulation, we first examined the relationship between this pathway and MDM2. As shown in figure 4E, MDM2 protein levels decreased in Panc-1 and Aspc-1 cells compared to control cells after treatment with curcumin, an inhibitor of the NF-κB signalling pathway. Conversely, MDM2 protein levels were higher in Panc-1 and Capan-2 cells than in control cells after treatment with increasing concentrations of TNF-α, a known activator of the NF-κB signalling pathway (figure 4F). To determine whether NF-κB signalling pathway activation is required for dysbindin-induced 10
MDM2 overexpression, we blocked the NF-κB signalling pathway with curcumin in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin cells) and control cells. We found that MDM2 protein expression levels did not change considerably in dysbindin-overexpressing cells compared with control cells (figure 4G). These findings indicate that the NF-κB signalling pathway can regulate MDM2 expression and that dysbindin-induced MDM2 overexpression is dependent on the NF-κB signalling pathway.
3.5 Dysbindin, a direct target of miR-342-3p, is negatively regulated by miR-342-3p in PDAC. Because dysbindin plays an important role in PDAC development, we wanted to determine how dysbindin is regulated in PDAC. MiRNAs have been reported to be closely involved in PDAC, but there is no published research on the relationship between miRNAs and dysbindin. Therefore, we aimed to elucidate whether miRNAs are involved in the regulation of dysbindin. We predicted candidate miRNAs targeting dysbindin using TargetScan software. As shown in figure 5A, we screened the top 5 miRNAs that potentially regulate dysbindin according to the context++ score generated by TargetScan software. Of these, two miRNAs had conserved target sites in the 3’UTR of dysbindin, and three had poorly conserved target sites. The predicted putative miRNA binding sites in the 3’UTR of dysbindin are shown in figure 5B. Based on these data, we transfected Aspc-1 cells with five candidate miRNA mimics (miR-199a-5p, miR-199b-5p, miR-182-5p, miR-377-3p and miR-342-3p). As shown in figure 5C, Western blotting showed that miR-342-3p had the most significant inhibitory effect on dysbindin. We used different cells (Aspc-1, Capan-2 and Bxpc-3) to validate that dysbindin protein levels were lower in PDAC cells transfected with miR-342-3p mimic than in control cells (figure 5D). Taken together, the data supported the selection of miR-342-3p for further experiments. Luciferase assays were performed to validate the direct binding of miR-342-3p to the 3′UTR of dysbindin. As shown in figure 5E, we identified three putative miR-342-3p binding sites in the 3’UTR of dysbindin mRNA. Luciferase assays showed that miR-342-3p overexpression significantly suppressed the WT dysbindin 3’UTR reporter construct but not the mutant construct. These data suggest that dysbindin is negatively regulated by miR-342-3p in PDAC and a direct target of miR-342-3p. 11
3.6 MiR-342-3p is downregulated in PDAC tissues, and dysbindin is significantly negatively correlated with miR-342-3p. The Cancer Genome Atlas (TCGA) data showed that in PDAC, miR-342-3p is downregulated (figure 6A), and dysbindin is upregulated (figure 6B). To validate miR-342-3p and dysbindin expression in PDAC, we measured miR-342-3p expression and dysbindin mRNA levels in 8 paired PDAC and adjacent noncancerous tissues by qRT-PCR. As shown in figure 6C, miR-342-3p expression was higher in adjacent noncancerous tissues than in PDAC tissues. Conversely, dysbindin expression was higher in PDAC tissues than in adjacent noncancerous tissues. Subsequently, we measured miR-342-3p expression in three PDAC cell lines (Panc-1, Bxpc-3 and Aspc-1) and a normal pancreatic ductal epithelial cell line (HPDE6c-7). As shown in figure 6D, miR-342-3p was downregulated in PDAC cell lines and upregulated in HPDE6c-7 cells. Dysbindin expression was significantly negatively correlated with miR-342-3p (figure 6E). In addition, TCGA data showed that low miR-342-3p expression levels were correlated with poor overall survival among PDAC patients (figure 6F). These data indicate that miR-342-3p is downregulated in PDAC and that dysbindin is significantly negatively correlated with miR-342-3p.
3.7 The antitumour effect of miR-342-3p on PDAC cell lines is decreased by dysbindin overexpression, and miR-342-3p decreases MDM2 expression. To determine the effect of miR-342-3p on PDAC metastasis and invasion, transwell assays were conducted with Aspc-1, Capan-2 and Panc-1 cells transfected with mimic control or miR-342-3p mimic. As shown in figure 7A, the miR-342-3p-transfected PDAC cells (Aspc-1, Capan-2 and Panc-1) were less metastatic and invasive than the control cells. We confirmed that miR-342-3p decreased dysbindin expression, and we next wondered whether dysbindin overexpression can reverse the antitumour effect of miR-342-3p in PDAC cell lines. Therefore, a dysbindin overexpression vector was constructed and transfected into Aspc-1 cells, and dysbindin expression levels in different groups are shown in figure 7B. Transwell assays showed that dysbindin overexpression significantly restored the migration and invasion capacities of 12
miR-342-3p-transfected cell lines (figure 7C). As the terminal molecule in the signalling axis, MDM2 plays a crucial role in NF-κB/MDM2 signalling, but it was unclear whether miR-342-3p affects MDM2 expression. Therefore, we detected MDM2 protein expression levels in PDAC cells (Panc-1 and Bxpc-3) transfected with mimic control or miR-342-3p mimic and found that MDM2 expression was lower in the miR-342-3p-transfected PDAC cell lines (Panc-1 and Bxpc-3) than in the control cells (figure 7D). In summary, these results show that the antitumour effect of miR-342-3p in PDAC cell lines is diminished by dysbindin overexpression and that miR-342-3p can decrease MDM2 expression.
4. Discussion Over the years, considerable efforts have been made to explore the molecular mechanism of PDAC invasion and metastasis; nonetheless, the mechanisms that lead to PDAC metastasis remain unknown. Our previous study reported that dysbindin expression is correlated with tumour size, tumour differentiation and clinical stage, indicating that dysbindin might be closely involved in PDAC metastasis. In this study, we found that dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. However, the precise mechanism by which dysbindin promotes PDAC metastasis remains unknown. To elucidate the potential molecular mechanism by which dysbindin promotes PDAC metastasis, we analysed differentially expressed cancer-related genes using a whole-transcript human gene expression array and selected MDM2 as a candidate gene. MDM2 is involved in the pathogenesis of different types of cancer, such as breast and cervical cancer[25, 26]. MDM2 is also related to tumour invasiveness in endometrial cancer[27]. In this study, we found that dysbindin overexpression increases MDM2 expression and that MDM2 knockdown decreases PDAC metastasis and invasion and attenuates the effects of dysbindin on these processes. These results suggest that dysbindin might promote PDAC cell invasion by upregulating MDM2. Therefore, we explored the potential signalling pathways involved in the regulation of MDM2 expression by dysbindin and found evidence for the potential participation of the NF-κB signalling pathway. We found that dysbindin upregulation increased key components of the NF-κB signalling pathway, indicating that dysbindin can activate this pathway. Dysbindin was reported to 13
promote NF-κB transcriptional activity in the nucleus in neuroblastoma cells[28], which is consistent with our results. When we blocked the NF-κB signalling pathway using curcumin, MDM2 expression was downregulated. In addition, we activated the NF-κB signalling pathway by upregulating TNF-α and MDM2 expression. These data show that dysbindin-induced MDM2 overexpression might depend on the NF-κB signalling pathway. Dysbindin plays a crucial role in PDAC metastasis. It is necessary to determine the underlying mechanism of dysbindin upregulation. A number of miRNAs are downregulated in PDAC and thus might regulate PDAC-related genes by affecting important signalling pathways[29]. In this study, miR-342-3p was identified as a candidate that potentially targets dysbindin. Recent studies have shown that miR-342-3p is downregulated in non-small cell lung cancer, nasopharyngeal carcinoma and cervical cancer[30-32]. We found that miR-342-3p expression negatively correlated with dysbindin expression and that dysbindin is a direct target of miR-342-3p. Rescue experiments demonstrated that the antitumour effect of miR-342-3p was decreased after dysbindin overexpression, indicating that dysbindin is a crucial target of miR-342-3p in PDAC. In conclusion, we demonstrated that dysbindin is regulated by miR-342-3p in PDAC and promotes PDAC metastasis and invasion by activating the NF-κB/MDM2 signalling axis. Thus, our findings clarify the mechanism of dysbindin upregulation in PDAC and how dysbindin promotes PDAC metastasis; ultimately, these data provide a therapeutic target for clinical intervention.
5. Acknowledgements This study was supported by grants from the National Key R&D Program of China (No. 2017YFC1308600), Key Research and Development Program of Shaanxi Province (No. 2017ZDXM-SF-024), and National Natural Science Foundation of China (No. 81670563). The authors are grateful for technical support from Mingzuo Jiang (State Key Laboratory of Cancer Biology, National Clinical Research Center for Digestive Diseases and Xijing Hospital of Digestive Diseases, Fourth Military Medical University) and Ning Xie (Department of Gastroenterology, the Second Affiliated Hospital of Xi’an Jiaotong University).
14
6. Conflict of interest The authors have declared that no conflicts of interest exist.
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Figure legends
Figure 1. Dysbindin promotes PDAC metastasis and invasion in vitro and in vivo. (A) Western blot analysis showing dysbindin expression in PDAC cells and human pancreatic ductal epithelial cells. The data are presented as the mean ± SEM. (B) Western blotting and qRT-PCR analysis showing dysbindin expression in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). The data are presented as the mean ± SEM. (C) After Panc-1 and Capan-2 cells were transfected with LV-shDysbindin (sh1 and sh2) or LV-shVector, Western blotting and qRT-PCR analysis were performed to measure dysbindin expression. The data are presented as the mean ± SEM. (D) Overexpression of dysbindin enhanced Aspc-1 and Bxpc-3 cell migration and invasion. The data are presented as the mean ± SEM. (E) Dysbindin knockdown decreased Panc-1 and Capan-2 cell migration and invasion. The data are presented as the mean ± SEM. (F) In vivo metastasis assays. Four stable cell lines (Aspc-1-LV-vector, Aspc-1-LV-dysbindin, Panc-1-LV-shvector and Panc-1-sh2dysbindin) were injected into the tail vein of nude mice. Representative H&E staining of the lungs from the above four groups is shown. Scale bars: top, 200 µm; bottom, 50 µm. (G, H) The incidence of lung metastasis and the number of metastatic lung nodules in nude mice from the above four groups after 8 weeks are shown. *P<0.05, **P<0.01, ***P<0.001.
Figure 2. Dysbindin overexpression increases MDM2 expression in PDAC, and MDM2 knockdown significantly attenuates dysbindin-enhanced metastasis and invasion. (A) A heatmap 19
was generated from the whole-transcript human gene expression array analysis of total RNA extracted from the Panc-1-dysbindin-siRNA and control groups. Red represents relatively lower expression, and green indicates relatively higher expression. (B) Western blot analysis showing MDM2 and dysbindin protein levels in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector). The data are presented as the mean ± SEM. (C) Western blot analysis showing MDM2 and dysbindin protein levels in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA) and control cells (Panc-1-control-siRNA and Capan-2-control-siRNA). The data are presented as the mean ± SEM. (D) After Capan-2 and Bxpc-3 cells were transfected with MDM2-siRNA or control-siRNA, Western blotting and qRT-PCR analysis were performed to determine MDM2 expression. The data are presented as the mean ± SEM. (E) MDM2 knockdown decreased Capan-2 and Bxpc-3 cell migration and invasion. The data are presented as the mean ± SEM. (F) After dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) were transfected with MDM2-siRNA or control-siRNA, Western blot analysis was performed to determine MDM2 and dysbindin protein levels. (G, H) MDM2 knockdown attenuated dysbindin-enhanced metastasis and invasion. The data are presented as the mean ± SEM. **P<0.01, ***P<0.001.
Figure 3. MDM2 expression is upregulated in PDAC tissues and correlates with clinicopathological characteristics, and dysbindin expression is positively correlated with MDM2 expression in PDAC tissues. (A) Western blot analysis showing MDM2 protein levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (B) qRT-PCR analysis showing MDM2 mRNA levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (C) MDM2 expression was detected on a tissue microarray containing 63 PDAC specimens and 57 adjacent noncancerous tissues. Representative IHC staining profiles of MDM2 in different groups are shown. ANT, adjacent noncancerous tissue; T, tumour tissue. Scale bars: top, 200 µm; bottom, 50 µm. (D) MDM2 expression levels in 63 PDAC tissues and 57 adjacent noncancerous tissues were analysed by Pearson's chi-square test. (E) Analysis of the IHC scores for MDM2 in PDAC 20
tissues and adjacent noncancerous tissues. ANT, adjacent noncancerous tissue; T, tumour tissue. The data are presented as the mean ± SEM. (F) Kaplan-Meier survival analysis showing the overall survival of PDAC patients with high or low MDM2 expression (n=63). (G) The association between dysbindin and MDM2 IHC scores in 20 PDAC tissues was analysed by Pearson’s correlation analysis (r=0.639, p<0.01). (H) Representative IHC staining profiles of dysbindin and MDM2 in PDAC tissues (n=20) and adjacent noncancerous tissues. Scale bars: top, 100 µm; bottom, 50 µm. ***P<0.001.
Figure 4. In PDAC cells, dysbindin activates the NF-κB signalling pathway, which regulates MDM2. Dysbindin-induced MDM2 overexpression is dependent on the NF-κB signalling pathway. (A) The top 20 significantly enriched biological processes identified by KEGG pathway analysis. The NF-κB signalling pathway was one of the most enriched pathways. (B-D) Western blot analysis showing the protein levels of NF-κB signalling pathway-related genes (IKKβ, p-IKKβ, IκBα, p-IκBα, p65 and p-p65) in dysbindin-knockdown cells (Panc-1-dysbindin-siRNA and Capan-2-dysbindin-siRNA), dysbindin-overexpressing cells (Bxpc-3-LV-dysbindin) and control cells. The data are presented as the mean ± SEM. (E) After PDAC cells (Panc-1 and Aspc-1) were treated with the NF-κB inhibitor curcumin (20 µM) for 24 h, Western blot analysis was performed to determine MDM2 and p-p65 protein levels in the curcumin-treated and DMSO (control)-treated groups. The data are presented as the mean ± SEM. (F) After PDAC cells (Panc-1 and Capan-2) were treated with control or increasing concentrations (10 or 20 ng/ml) of TNF-α, an NF-κB signalling pathway activator, Western blot analysis was performed to determine MDM2 and p-p65 protein levels in each group. The data are presented as the mean ± SEM. (G) Western blot analysis showing MDM2 and p-p65 protein levels in dysbindin-overexpressing cells (Aspc-1-LV-dysbindin and Bxpc-3-LV-dysbindin) and control cells (Aspc-1-LV-vector and Bxpc-3-LV-vector) treated with curcumin (20 µM) for 24 h. The data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 5. Dysbindin, a direct target of miR-342-3p, is negatively regulated by miR-342-3p in PDAC. (A) The top 5 predicted candidate miRNAs (miR-199a-5p, miR-199b-5p, miR-182-5p, 21
miR-377-3p and miR-342-3p) targeting dysbindin according to TargetScan software. (B) Predicted pairing to the target region in the 3’UTR of dysbindin (top) and the five predicted candidate miRNAs (bottom). (C) Western blot analysis showing dysbindin protein levels in Aspc-1 cells transfected with the five candidate miRNA mimics (miR-199a-5p, miR-199b-5p, miR-182-5p, miR-377-3p and miR-342-3p). The data are presented as the mean ± SEM. (D) After PDAC cells (Aspc-1, Capan-2 and Bxpc-3) were transfected with miR-342-3p mimic or mimic control, Western blot analysis was performed to determine dysbindin protein levels. The data are presented as the mean ± SEM. (E) Three predicted binding sites of miR-342-3p in the 3’UTR of dysbindin mRNA. MiR-342-3p decreased the luciferase activity of the WT dysbindin reporter but not the mutant reporter. The data are presented as the mean ± SEM. *P<0.05, **P<0.01, ***P<0.001.
Figure 6. MiR-342-3p is downregulated in PDAC, and dysbindin is significantly negatively correlated with miR-342-3p. (A) MiR-342-3p is downregulated in PDAC according to The Cancer Genome Atlas (TCGA) data. NP, normal pancreas tissue; PC, pancreatic cancer tissue. (B) Dysbindin expression is upregulated in PDAC according to TCGA data. N, noncancerous tissue; T, tumour tissue. (C) qRT-PCR analysis showing miR-342-3p and dysbindin mRNA levels in PDAC tissues (n=8) and paired adjacent noncancerous tissues. The data are presented as the mean ± SEM. (D) qRT-PCR analysis showing miR-342-3p expression levels in PDAC cell lines (Panc-1, Bxpc-3 and Aspc-1) and a normal pancreatic ductal epithelial cell line (HPDE6c-7). The data are presented as the mean ± SEM. (E) The association between dysbindin mRNA and miR-342-3p expression was analysed by Pearson’s correlation coefficient (r=-0.6685, p<0.01). (F) Kaplan-Meier survival analysis showing the overall survival of PDAC patients with high or low miR-342-3p expression from TCGA data. *P<0.05, **P<0.01, ***P<0.001.
Figure 7. The antitumour effect of miR-342-3p on PDAC cell lines is diminished by dysbindin overexpression, and miR-342-3p decreases MDM2 expression. (A) MiR-342-3p-transfected PDAC cells (Aspc-1, Capan-2 and Panc-1) were less metastatic and invasive than control cells. The data are presented as the mean ± SEM. (B) Western blot analysis showing dysbindin protein 22
levels in the Aspc-1-vector, Aspc-1-miR-342-mimic and Aspc-1-miR-342-mimic+dysbindin groups. The data are presented as the mean ± SEM. (C) Transwell assays showing that dysbindin overexpression reverses the ability of miR-342-3p to inhibit PDAC cell (Aspc-1) migration and invasion. The data are presented as the mean ± SEM. (D) Western blot analysis showing MDM2 expression in miR-342-3p-transfected PDAC cells (Panc-1 and Bxpc-3) and control cells. The data are presented as the mean ± SEM. (E) Proposed model by which the NF-κB/MDM2 signalling axis is activated by dysbindin, which is regulated by miR-342-3p in PDAC cells. *P<0.05, **P<0.01, ***P<0.001.
23
Table1. Correlations between MDM2 expression and clinical characteristics in patients with PDAC. Variable
Patient number (n=63)
MDM2 expression high
low
(n=34)
(n=29)
P-value (χ2 test)
Age (years) ≤60
24
15
9
＞60
39
19
20
Male
36
24
12
Female
27
10
17
Head
37
20
17
Body/tail
26
14
12
≤4
42
21
21
＞4
21
13
8
N0
35
16
19
N1
25
15
10
Unknown
3
0.191
Gender
0.06
Tumor location
0.506
Tumor size (cm)
0.000* .000*
Nodal status
Perineural invasion
0.423
Absent
36
20
16
Present
27
14
13
Ⅰ,Ⅱ
34
15
19
Ⅲ
29
19
10
0.609
Histological Grade (differentiation)
*Statistically significant. PP-value < 0.05 are in bold.
0.000* .000*
Table2. Univariate and multivariate analyses of prognostic parameters for survival in PDAC patients. Variable
Univariate analysis HR
95% CI
P-value
MDM2 expression (high vs. low)
2.085
1.175-3.699
0.012*
Age (≤ (≤60 years vs. >60 years) years)
1.278
0.721- 2.268
0.401
Gender (male vs. female) female)
0.878
0.5-1.541
0.651
Tumor location (head vs. body/tail)
1.229
0.7-2.157
0.473
Tumor size (≤4 cm vs. >4 >4 cm)
1.589
0.873-2.895
0.13
Nodal status(N0 status(N0 vs. N1)
1.479
0.841-2.6
0.174
Perineural invasion(absent invasion(absent vs. present)
0.434
0.242-0.78
0.005*
Histological Grade (differentiation)
1.425
0.816-2.489
0.213
Variable
Multivariate analysis HR
95% CI
P-value
MDM2 expression (high vs. low)
2.523
1.276-4.987
0.008*
Perineural invasion(absent invasion(absent vs. present)
0.337
0.167-0.681
0.002*
Univariate analysis and multivariate analyses were analyzed by Cox proportional hazards model. HR, hazard ratio; 95% CI, 95% confidence interval. *Statistically significant. P-value < 0.05 are in bold.
1. Dysbindin could promote PDAC metastasis and invasion in vitro and in vivo. 2. Dysbindin could promote metastasis of PDAC via NF-κB/MDM2 signal axis. 3. MDM2 is upregulated in PDAC tissues and correlated with clinicopathological characteristics of PDAC. 4. Dysbindin is a direct target of miR-342-3p. 5. Dysbindin overexpression could decrease the antitumor effect of miR-342-3p.
The authors have declared that no conflict interests exist.
Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most invasive solid tumours and has the highest cancer-related mortality rate. Despite intense investigation, the molecular mechanisms underlying the invasiveness and aetiology of PDAC remain elusive. MicroRNAs (miRNAs) are key regulators of tumour cell plasticity, but their roles in PDAC metastasis have not been characterized. Our early studies showed that dysbindin protein levels are elevated in PDAC patients compared with control individuals and that dysbindin upregulation elicits PDAC cell proliferation via the PI3K pathway. Here, we show that dysbindin promoted PDAC metastasis via the NF-κB/MDM2 signalling axis. Increased dysbindin levels correlated with aggressive features in PDAC, and the overexpression of dysbindin significantly promoted PDAC metastasis and invasion in vitro and in vivo. Surprisingly, dysbindin was identified as a direct target of miR-342-3p, which promotes NF-κB activation and PDAC metastasis. Thus, dysbindin-mediated NF-κB activation via miR-342-3p represents a context-dependent switch that enables PDAC cell proliferation and metastasis. Our data suggest that dysbindin and miR-342-3p are potential leads for the development of targeted therapy for PDAC.
Keywords: Dysbindin, miR-342-3p, Pancreatic ductal adenocarcinoma, NF-κB/MDM2, metastasis