Background/Aims: Mounting evidence suggests that epitranscriptional modifications regulate multiple cellular processes. N⁶-Methyladenosine (m⁶A), the most abundant reversible methylation of mRNA, has critical roles in cancer pathogenesis. However, the mechanisms and functions of long non-coding RNA (lncRNA) methylation remain unclear. Pancreatic cancer resulted in 411,600 deaths globally in 2015. By the time of pancreatic cancer diagnosis, metastasis has often occurred in other parts of the body. The present study sought to investigate lncRNA m⁶A modification and its roles in pancreatic cancer. Methods: Differential expression between cancer cells and matched normal cells was evaluated to identify candidate lncRNAs. The lncRNA KCNK15-AS1 was detected in cancer tissues and various pancreatic cells using RT-qPCR. KCNK15-AS1 was transfected into cells to explore its role in migration and invasion. Then, m⁶A RNA immunoprecipitation was performed to detect methylated KCNK15-AS1 in tissues and cells. Epithelial–mesenchymal transition (EMT) markers were used to evaluate KCNK15-AS1-mediated EMT processes. Results: KCNK15-AS1 was downregulated in pancreatic cancer tissues compared with paired adjacent normal tissues. KCNK15-AS1 inhibited migration and invasion in MIA PaCa-2 and BxPC-3 cells. Furthermore, total RNA methylation in cancer cells was significantly enriched relative to that in immortalized human pancreatic duct epithelial (HPDE6-C7) cells. In addition, the m⁶A eraser ALKBH5 was downregulated in cancer cells, which can demethylate KCNK15-AS1 and regulate KCNK15-AS1-mediated cell motility. Conclusion: Our results have revealed a novel mechanism by which ALKBH5 inhibits pancreatic cancer motility by demethylating lncRNA KCNK15-AS1, identifying a potential therapeutic target for pancreatic cancer.

Galectin-1 is implicated in making tumor cells immune privileged, in part by regulating the survival of infiltrating T cells. Galectin-1 is strongly expressed in activated pancreatic stellate cells (PSCs); however, whether this is linked to tumor cell immune escape in pancreatic cancer is unknown. Galectin-1 was knocked down in PSCs isolated from pancreatic tissues using small interfering RNA (siRNA), or overexpressed using recombinant lentiviruses, and the PSCs were cocultured with T cells. CD31 , CD4 1 and CD8 1 T cell apoptosis was detected by flow cytometry; T cell IL-2, IL-4, IL-5 and INF-c production levels were quantified using ELISA. Immunohistochemical analysis showed increased numbers of PSCs expressed Galectin-1 (p < 0.01) and pancreatic cancers had increased CD3 1 T cell densities (p < 0.01) compared to normal pancreas or chronic pancreatitis samples. In coculture experiments, PSCs that overexpressed Galectin-1 induced apoptosis of CD4 1 T cells (p < 0.01) and 05). Supernatants from T cells cocultured with PSCs that overexpressedGalectin-1 contained significantly increased levels of Th2 cytokines (IL-4 and IL-5, p < 0.01) and decreased Th1 cytokines (IL-2 and INF-c, p < 0.01). However, the knockdown of PSC Galectin-1 had the opposite effect on Th1 and Th2 cytokine secretion. Our study suggests that the overexpression of Galectin-1 in PSCs induced T cell apoptosis and Th2 cytokine secretion, which may regulate PSC-dependent immunoprivilege in the pancreatic cancer microenvironment. Galectin-1 may provide a novel candidate target for pancreatic cancer immunotherapy.Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive malignancy, which is resistant to currently available systemic therapies. PDAC has one of the worst prognoses of all human cancers with incidence rates nearly equal to mortality rates. 1 There is evidence that excessive desmoplasia play a crucial role in the aggressive behavior of pancreatic cancer, 2 which impedes effective systemic treatments on a molecular level. Pancreatic stellate cells (PSCs) are stellate-shaped mesenchymal pancreatic cells, which have been identified as important regulators of desmoplasia in PDAC. 3In their quiescent state, PSCs can be identified by the presence of vitamin A-containing lipid droplets in the cytoplasm and the expression of desmin and glial-fibrillary-acidic protein (GFAP). 4 In response to pancreatic damage or stress, PSC are transformed into an activated myofibroblast-like phenotype. Activated PSCs express a-smooth muscle actin (a-SMA), and synthesize excessive amounts of ECM proteins, including Collagen I and III, fibronectin and matrix-degrading enzymes such as MMPs.5-7 Activated PSCs have a variety of cell functions and can promote the proliferation, migration, invasion and metastasis of pancreatic cancer cells; 1,8,9 however, the factors that PSCs secrete to advance pancreatic cancer progression remain largely unknown.Galectin-1, a member of the galectin family of b-galactosidebinding proteins, is a homodimer of 14-kDa subunits pos...

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Pancreatic ductal adenocarcinoma (PDAC) is one of the most common malignant tumors with poor prognosis due to extremely high malignancy, low rate of eligibility for surgical resection and chemoradiation resistance. Increasing evidence indicate that the interaction between activated pancreatic stellate cells (PSCs) and PDAC cells plays an important role in the development of PDAC. By producing high levels of cytokines, chemotactic factors, growth factors and excessive extracellular matrix (ECM), PSCs create desmoplasia and a hypoxic microenvironment that promote the initiation, development, evasion of immune surveillance, invasion, metastasis and resistance to chemoradiation of PDAC. Therefore, targeting the interaction between PSCs and PDAC cells may represent a novel therapeutic approach to advanced PDAC, especially therapies that target PSCs of the pancreatic tumor microenvironment.Pancreatic ductal adenocarcinoma (PDAC) is the most common and lethal malignant tumors in humans with very poor prognosis, due to a variety of causes including the insidious onset, absence of efficient screening methods for early detection, low rate of surgical resection at the time of clinical presentation and chemoradiation resistance. 1 Although scholars have been committed during the last three decades to studying genetic and/or epigenetic molecular changes of PDAC cells and chemotherapy with a combination of cytotoxic drugs have been implemented, the survival of patients with PDAC has not yet significantly improved.2,3 Until recently, an encouraging FOLFIRINOX scheme was reported to provide a statistically and clinically significant benefit over single-agent gemcitabine in patients with advanced PDAC but Key words: pancreatic ductal adenocarcinoma, pancreatic stellate cells, tumor microenvironment Abbreviations: ADMR: adrenomedullin receptor; AM: adrenomedullin; ATRA: all-trans retinoic acid; CTGF: connective tissue growth factor; DCs: dendritic cells; ECM: extracellular matrix; EGF: epidermal growth factor; EMMPRIN: extracellular matrix metalloproteinase inducer; EMT: epithelial-mesenchymal transition; ET-1: endothelin-1; FGF: fibroblast-growth-factor; GFAP: glial-fibrillary-acidic protein; HGF: hepatocyte growth factor; HPDE: human pancreatic duct epithelial; HSCs: hepatic stellate cells; HUVEC: endothelial cells; IGF-1: insulin-like growth factor-1; IL: interleukin; MMPs: matrix metalloproteinases; NADPH: nicotinamide adenine dinucleotide phosphate; NO: nitric oxide; PAEE: palmitic acid ethyl ester; PCLMs: pancreatic cancer liver metastases; PDAC: pancreatic ductal adenocarcinoma; PDGF: platelet-derived growth factor; PEDF: pigment epithelium-derived factor; PK: prokineticin; PKR: prokineticin receptor; PSCs: pancreatic stellate cells; ROS: oxygen species; Runx-2: Runt-related transcription factor-2; SDF-1: stromal cell-derived factor-1; sFRP4: secreted frizzled-related protein 4; SMO: smoothened; a-SMA: a-smooth muscle actin; SPARC: secreted protein acidic and rich in cysteine; TFF1: trefoil factor 1; TGF-b: transforming gr...

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Background A growing number of studies have focused on investigating circRNAs as crucial regulators in the progression of multiple cancer types. Nevertheless, the biological effects and underlying mechanisms of circRNAs in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Methods Differentially expressed circRNAs between cancerous tissue and adjacent normal tissues were identified by RNA sequencing in PDAC. Subsequently, in vitro and in vivo functional experiments were performed to investigate the functional roles of circNEIL3 in PDAC tumour growth and metastasis. Furthermore, RNA pull-down, dual-luciferase reporter assays, RNA immunoprecipitation (RIP) assays, fluorescent in situ hybridization (FISH) and Sanger sequencing assays were performed to examine the circular interaction among circNEIL3, miR-432-5p and adenosine deaminases acting on RNA 1 (ADAR1). Results CircNEIL3 was upregulated in PDAC and promoted the progression of PDAC cells both in vitro and in vivo. Mechanistically, circNEIL3 was shown to regulate the expression of ADAR1 by sponging miR-432-5p to induce RNA editing of glioma-associated oncogene 1 (GLI1), ultimately influencing cell cycle progression and promoting epithelial-to-mesenchymal transition (EMT) in PDAC cells. Moreover, we discovered that the circNEIL3/miR-432-5p/ADAR1 axis was correlated with the PDAC clinical stage and overall survival of PDAC patients, while ADAR1 may reduce the biogenesis of circNEIL3. Conclusions Our findings reveal that circNEIL3 facilitates the proliferation and metastasis of PDAC through the circNEIL3/miR-432-5p/ADAR1/GLI1/cell cycle and EMT axis and that its expression is regulated by ADAR1 through a negative feedback loop. Therefore, circNEIL3 may serve as a prognostic marker and a therapeutic target for PDAC.

BackgroundNatural killer (NK) cells play a key role in non-specific immune response in different cancers, including pancreatic cancer. However the anti-tumor effect of NK cells decreases during pancreatic cancer progression. The regulatory pathways by which NK cells facilitate tumor immune escape are unclear, therefore our purpose was to investigate the roles of the contributory factors.MethodsNK cells isolated from fresh healthy peripheral blood were co-cultured with normal human pancreatic ductal cells hTERT-HPNE and human pancreatic cancer cell lines SW1990 and BxPc-3 in vitro. Then NK cell function was determined by Flow cytometric analysis of surface receptors and cytotoxic granules in NK cells, NK cell apoptosis and cytotoxicity, and Enzyme-linked immunosorbent assay of cytokines. Expression level of MMP-9, IDO and COX-2 in hTERT-HPNE and SW1990 cells were detected by quantitative RT-PCR. Statistical differences between data groups were determined by independent t-tests using SPSS 19.0 software.ResultsOur results showed that NK cell function was significantly downregulated following exposure to pancreatic cancer cells compared to normal pancreatic cells, as demonstrated by lower expressions of activating surface receptors (NKG2D, DNAM-1, NKp30 and NKp46) and cytotoxic granules (Perforin and Granzyme B); decreased secretion of cytokines (TNF-α and IFN-γ); and reduced cytotoxicity against myelogenous leukemia K562 cells. Further investigations revealed that MMP-9 and IDO may be implicated in SW1990 cell-induced NK cell dysfunction by facilitating tumor immune evasion. Blockade by TIMP-1 and/or 1-MT could partially restore NK function.ConclusionsTaken together, elevation of MMP-9 and IDO induced by pancreatic cancer cells mediates NK cell dysfunction. Our findings could contribute to the development of NK cell-based immunotherapy in patients with pancreatic cancer.

BackgroundLong non-coding RNAs (lncRNAs) play an important role in the development and progression of various tumors, including pancreatic cancer (PC). Recent studies have shown that lncRNAs can ‘act in cis’ to regulate the expression of its neighboring genes. Previously, we used lncRNAs microarray to identify a novel lncRNA termed XLOC_000647 that was down-regulated in PC tissues. However, the expression and function of XLOC_000647 in PC remain unclear.MethodsThe expression of XLOC_000647 and NLRP3 in PC specimens and cell lines were detected by quantitative real-time PCR. Transwell assays were used to determine migration and invasion of PC cells. Western blot was carried out for detection of epithelial-mesenchymal transition (EMT) markers in PC cells. The effect of XLOC_000647 on PC cells was assessed in vitro and in vivo. The function of NOD-like receptor family pyrin domain-containing 3 (NLRP3) in PC was investigated in vitro. In addition, the regulation of NLRP3 by XLOC_000647 in PC was examined in vitro.ResultsHere, XLOC_000647 expression was down-regulated in PC tissues and cell lines. The expression level of XLOC_000647 was significantly correlated to tumor stage, lymph node metastasis, and overall survival. Overexpression of XLOC_000647 attenuated cell proliferation, invasion, and EMT in vitro and impaired tumor growth in vivo. Further, a significantly negative correlation was observed between XLOC_000647 levels and its genomic nearby gene NLRP3 in vitro and in vivo. Moreover, XLOC_000647 decreased NLRP3 by inhibiting its promoter activity. Knockdown of NLRP3 decreased proliferation of cancer cells, invasion, and EMT in vitro. Importantly, after XLOC_000647 was overexpressed, the corresponding phenotypes of cells invasion and EMT were reversed by overexpression of NLRP3.ConclusionsTogether, these results indicate that XLOC_000647 functions as a novel tumor suppressor of lncRNA and acts as an important regulator of NLRP3, inhibiting cell proliferation, invasion, and EMT in PC.