Background N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). Methods The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. Results ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. Conclusion ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy.

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Induced pluripotent stem cells (iPSCs) have the potential to revolutionise cell therapy; however, it remains unclear whether iPSCs can be generated from human osteoarthritic chondrocytes (OCs) and subsequently induced to differentiate into chondrocytes. In the present study, we investigated the differentiation potential of OCs into iPSCs using defined transcription factors and explored the possibility of using these OC-derived iPSCs for chondrogenesis. Our study demonstrates that iPSCs can be generated from OCs and that these iPSCs are indistinguishable from human embryonic stem cells (hESCs). To promote chondrogenic differentiation, we used lentivirus to transduce iPSCs seeded in alginate matrix with transforming growth factor-β1 (TGF-β1) and then in vitro co-cultured these iPSCs with chondrocytes. Gene expression analysis showed that this combinational strategy promotes the differentiation of the established iPSCs into chondrocytes in alginate matrix. Increased expression of cartilage-related genes, including collagen II, aggrecan, and cartilage oligomeric matrix protein (COMP), and decreased gene expression of the degenerative cartilage marker, vascular endothelial growth factor (VEGF), were observed. The histological results revealed a dense sulphated extracellular matrix in the co-culture of TGF-β1-transfected iPSCs with chondrocytes in alginate matrix. Additionally, in vivo chondroinductive activity was also evaluated. Histological examination revealed that more new cartilage was formed in the co-culture of TGF-β1-transfected iPSCs with chondrocytes in alginate matrix. Taken together, our data indicate that iPSCs can be generated from OCs by defi ned factors and the combinational strategy results in significantly improved chondrogenesis of OC-derived iPSCs. This work adds to our understanding of potential solutions to osteoarthritic cell replacement problem.

Junctional adhesion molecule-C (JAM-C) plays a key role in the promotion of the reverse transendothelial migration (rTEM) of neutrophils, which contributes to the dissemination of systemic inflammation and to secondary organ damage. During acute pancreatitis (AP), systemic inflammatory responses lead to distant organ damage and typically result in acute lung injury (ALI). Here, we investigated the role of rTEM neutrophils in AP-associated ALI and the molecular mechanisms by which JAM-C regulates neutrophil rTEM in this disorder. In this study, rTEM neutrophils were identified in the peripheral blood both in murine model of AP and human patients with AP, which elevated with increased severity of lung injury. Pancreatic JAM-C was downregulated during murine experimental pancreatitis, whose expression levels were inversely correlated with both increased neutrophil rTEM and severity of lung injury. Knockout of JAM-C resulted in more severe lung injury and systemic inflammation. Significantly greater numbers of rTEM neutrophils were present both in the circulation and pulmonary vascular washout in JAM-C knockout mice with AP. This study demonstrates that during AP, neutrophils that are recruited to the pancreas may migrate back into the circulation and then contribute to ALI. JAM-C downregulation may contribute to AP-associated ALI via promoting neutrophil rTEM.

D receptors control pancreatic inflammation in AP by inhibiting NF-κB activation via a PP2A-dependent Akt signalling pathway.

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BackgroundPrevious studies have investigated the sustained aberrantly activated Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is crucial for pancreatic cancer growth and metastasis. Suppressor of cytokine signaling 3 (SOCS3), as a key negative feedback regulator of this signaling pathway, is usually down-regulated in various cancers. In the present study, we aim at exploring the biological function and the underlying molecular regulation mechanisms of SOCS3 in pancreatic cancer.MethodsThe expression of SOCS3 and other genes in pancreatic cancer was examined by Quantitative real-time PCR, western blotting and immunohistochemical staining. The interaction between pSTAT3 and DNA Methyltransferase 1 (DNMT1) was investigated by co-immunoprecipitation assay. Luciferase reporter assay was used to investigate the transcriptional regulation of pSTAT3 and DNMT1 on the SOCS3 gene. The effects of SOCS3 on the biological behavior of pancreatic cancer cells were assessed both in vitro and vivo. Furthermore, we performed a comprehensive analysis of the expression of SOCS3 in a pancreatic cancer tissue microarray (TMA) and correlated our findings with pathological parameters and outcomes of the patients.ResultsWe showed that SOCS3 expression was decreased in phosphorylated STAT3 (pSTAT3)-positive tumors and was negatively correlated with pSTAT3 in pancreatic cancer cells. We also found that IL-6/STAT3 promoted SOCS3 promoter hypermethylation by increasing DNMT1 activity; silencing DNMT1 or 5-aza-2-deoxycytidine (5-AZA) treatment could reverse the down-regulation of SOCS3 mediated by IL-6. Using co-immunoprecipitation and luciferase reporter assays, we found that STAT3 recruited DNMT1 to the promoter region of SOCS3 and inhibited its transcriptional activity. Overexpression of SOCS3 significantly inhibited cell proliferation, which may be due to the increase in G1-S phase arrest; overexpression of SOCS3 also inhibited cell migration and invasion as well as tumorigenicity in nude mice. Pancreatic cancer tissue microarray analysis showed that high SOCS3 expression was a good prognostic factor and negatively correlated with tumor volume and metastasis.ConclusionWe demonstrated that activated IL-6/STAT3 signaling could induce SOCS3 methylation via DNMT1, which led to pancreatic cancer growth and metastasis. These data also provided a mechanistic link between sustained aberrantly activated IL-6/STAT3 signaling and SOCS3 down-regulation in pancreatic cancer. Thus, inhibitors of STAT3 or DNMT1 may become novel strategies for treating pancreatic cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0301-7) contains supplementary material, which is available to authorized users.

Background and AimsGLI1, as an indispensable transcriptional factor of Hedgehog signaling pathway, plays an important role in the development of pancreatic cancer (PC). DNA methyltransferases (DNMTs) mediate the methylation of quantity of tumor-related genes. Our study aimed to explore the relationship between GLI1 and DNMTs.MethodsExpressions of GLI1 and DNMTs were detected in tumor and adjacent normal tissues of PC patients by immunohistochemistry (IHC). PANC-1 cells were treated by cyclopamine and GLI1-siRNA, while BxPC-3 cells were transfected with overexpression-GLI1 lentiviral vector. Then GLI1 and DNMTs expression were analyzed by qRT-PCR and western blot (WB). Then we took chromatin immunoprecipitation (ChIP) to demonstrate GLI1 bind to DNMT1. Finally, nested MSP was taken to valuate the methylation levels of APC and hMLH1, when GLI1 expression altered.ResultsIHC result suggested the expressions of GLI1, DNMT1 and DNMT3a in PC tissues were all higher than those in adjacent normal tissues (p<0.05). After GLI1 expression repressed by cyclopamine in mRNA and protein level (down-regulation 88.1±2.2%, 86.4±2.2%, respectively), DNMT1 and DNMT3a mRNA and protein level decreased by 91.6%±2.2% and 83.8±4.8%, 87.4±2.7% and 84.4±1.3%, respectively. When further knocked down the expression of GLI1 by siRNA (mRNA decreased by 88.6±2.1%, protein decreased by 63.5±4.5%), DNMT1 and DNMT3a mRNA decreased by 80.9±2.3% and 78.6±3.8% and protein decreased by 64.8±2.8% and 67.5±5.6%, respectively. Over-expression of GLI1 by GLI1 gene transfection (mRNA increased by 655.5±85.9%, and protein increased by 272.3±14.4%.), DNMT1 and DNMT3a mRNA and protein increased by 293.0±14.8% and 578.3±58.5%, 143.5±17.4% and 214.0±18.9%, respectively. ChIP assays showed GLI1 protein bound to DNMT1 but not to DNMT3a. Results of nested MSP demonstrated GLI1 expression affected the DNA methylation level of APC but not hMLH1 in PC.ConclusionDNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.