Microarray-based gene expression profiling reveals genes and pathways involved in the oncogenic function of REG3A on pancreatic cancer cells.

We previously reported that regenerating islet-derived protein 3 alpha (REG3A) exacerbates pancreatic malignancies. The mechanism of this effect has not been clearly elucidated. Here we first identified key differentially expressed genes (DEGs) and signal pathways in the pancreatic cancer cell line SW1990, compared to two control cell lines, by microarray analysis. We then identified key genes and pathways regulated by REG3A or the cytokine IL6 in SW1990 cells. Afterwards, these DEGs induced by REG3A or IL6 were subjected to KEGG pathway enrichment analysis and GO function analysis by the DAVID online tool. Ultimately, we constructed protein-protein interaction networks among the DEGs by Cytoscape. Among the three pancreatic cell lines, SW1990 exhibited highly deterioration with the activation of genes and pathways related to proliferation, survival, angiogenesis, and invasion. As a result, 50 DEGs enriched in 11 pathways were identified in REG3A-treated SW1990 cells, and 28 DEGs enriched in 9 pathways were detected in IL6-treated cells. Overall, results of microarray analysis followed by qRT-PCR and Western blotting suggest that REG3A regulates pancreatic cell growth by increasing the expression of at least 8 genes: JAK1, STAT3, IL10, FOXM1, KRAS, MYC, CyclinD1, and c-fos; and activation of at least 4 signal pathways: TGFß, PDGF, angiogenesis and RAS. Similar results were obtained with IL6 treatment. Regulation network analysis confirmed the cell growth related DEGs, and further uncovered three transcription factor families with immune functions regulated by REG3A.

Reg3g Promotes Pancreatic Carcinogenesis in a Murine Model of Chronic Pancreatitis.

BACKGROUND: Regenerating islet-derived 3 (Reg3) is abnormally expressed in several human digestive system diseases, including chronic pancreatitis (CP) and pancreatic cancer (PC). AIM: The purpose of this study was to clarify the mechanism of the enhanced expression of Reg3 in inflammation-induced PC. METHODS: C57BL/6 mice were treated with caerulein for 6 weeks to induce CP and then injected with pReg3g--a lentivirus system encoding for murine Reg3g--accompanied by dimethylbenzanthracene to induce PC. We detected pancreatic histopathological characteristics, tumor-related gene expression, inflammation-associated pathway activation, serum biochemical indicators, and immunological cell activities. RESULTS: The mice that developed CP after caerulein treatment were marked by pronounced histologic lesions, elevated serum amylase levels, and activation of inflammation-related pathways. Mice given a high dose of pReg3g developed PC by 16 weeks, with recognizable tumors in the pancreas. While, both the low and high doses of pReg3g produced higher transcription of c-fos, k-ras, cytokeratin-19, and proliferating cell nuclear antigen, and a lower expression of caspase-3 compared to pNEG controls. Additionally, the higher dose of pReg3g increased the expressions of pSTAT3, NFκB (p65), and SOCS3 methylation during PC development. In addition, mice treated with pReg3g displayed higher levels of serum IL10 and TGFß and suppressed T lymphocyte proliferation and DC function. CONCLUSION: The comprehensive analysis suggests enhanced Reg3g expression exacerbates PC in inflammation-associated cancer progression. Reg3g appears to promote CP-related PC in mice through multiple mechanisms, involving enhanced transcription of pancreatic tumor markers, repression of anti-tumor immunity, and activation of STAT3/p65 signal transduction pathways.

Multiple mechanisms involved in diabetes protection by lipopolysaccharide in non-obese diabetic mice.

Toll-like receptor 4 (TLR4) activation has been proposed to be important for islet cell inflammation and eventually ß cell loss in the course of type 1 diabetes (T1D) development. However, according to the "hygiene hypothesis", bacterial endotoxin lipopolysaccharide (LPS), an agonist on TLR4, inhibits T1D progression. Here we investigated possible mechanisms for the protective effect of LPS on T1D development in non-obese diabetic (NOD) mice. We found that LPS administration to NOD mice during the prediabetic state neither prevented nor reversed insulitis, but delayed the onset and decreased the incidence of diabetes, and that a multiple-injection protocol is more effective than a single LPS intervention. Further, LPS administration suppressed spleen T lymphocyte proliferation, increased the generation of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs), reduced the synthesis of strong Th1 proinflammatory cytokines, and downregulated TLR4 and its downstream MyD88-dependent signaling pathway. Most importantly, multiple injections of LPS induced a potential tolerogenic dendritic cell (DC) subset with low TLR4 expression without influencing the DC phenotype. Explanting DCs from repeated LPS-treated NOD mice into NOD/SCID diabetic mice conferred sustained protective effects against the progression of diabetes in the recipients. Overall, these results suggest that multiple mechanisms are involved in the protective effects of LPS against the development of diabetes in NOD diabetic mice. These include Treg induction, down-regulation of TLR4 and its downstream MyD88-dependent signaling pathway, and the emergence of a potential tolerogenic DC subset.

REG3A accelerates pancreatic cancer cell growth under IL-6-associated inflammatory condition: Involvement of a REG3A-JAK2/STAT3 positive feedback loop.

Regenerating gene protein (REG) 3A is a 19 kD secretory pancreas protein with pro-growth function. Previously we demonstrated that overexpression of REG3A, acting as a key molecule for up-regulation of the JAK2/STAT3 pathway, contributed to inflammation-related pancreatic cancer (PaC) development. However the exact network associated with REG3A signaling still remains unclear. Here we determined that exposure of human PaC cells to cytokine IL-6 activated the oncogenic JAK2/STAT3 pathway, which directly upregulated REG3A expression, accelerated cell cycle progression by promoting CyclinD1 expression, and enhancing the expression of the anti-apoptosis Bcl family. Importantly, the activation of REG3A would instead enhance the JAK2/STAT3 pathway to constitute a REG3A-JAK2/STAT3 positive feedback loop, which leads to the amplification of the oncogenic effects of IL-6/JAK2/STAT3, a classic pathway linking to inflammation-related tumorigenesis, ultimately resulting in PaC cell over-proliferation and tumor formation both in vitro and in vivo. Moreover, EGFR was found to mediate the REG3A signal for PaC cell growth and JAK2/STAT3 activation, thus functioning as a REG3A receptor. Collectively, our results provide the first evidence for the presence of the synergistic effect of REG3A and IL-6 on PaC development via a REG3A-JAK2/STAT3 positive feedback loop.

SOCS3 methylation in synergy with Reg3A overexpression promotes cell growth in pancreatic cancer.

UNLABELLED: Pancreatic cancer (PaC) is the fifth leading cause of cancer death in the world, but the molecular mechanisms for its development remain unclear. Regenerating islet-derived protein 3-alpha (Reg3A) has been reported overexpressed in pancreatic inflammation and associated with PaC malignancies, thus believed as a potential target in inflammation-linked pancreatic carcinogenesis. Silencing of suppressor of cytokine signaling SOCS3, a well-known feedback inhibitor of cell proliferation, has been found in many human cancers. Here, we identified that SOCS3 was aberrantly methylated in its CpG island in 3/5 human PaC cell lines and 11/36 cancer tissue samples. SOCS3 restoration by a demethylating agent, 5-aza-2'-deoxycytidine, remarkably suppressed cell proliferation and induced apoptosis of methylated PaC cells. Moreover, we also have shown that Reg3A was highly expressed in PaC cells and tissue samples. Assessment of potential relationship between SOCS3 and Reg3A aberrations in vitro revealed that SOCS3 worked downstream of Reg3A and modulated Reg3A-linked pro-tumor functions. siRNA-mediated SOCS3 knock-down in normal pancreatic epithelial cells and plasmid-transfected SOCS3 overexpression in PaC cells, respectively, resulted in the obvious promotion and inhibition of Reg3A-induced cell proliferation, thereby suggesting SOCS3 negatively regulating Reg3A-mediated PaC progression. In addition, our findings also revealed that JAK/STAT3/NF-κB appear involved in the effect of SOCS3-Reg3A interaction on pancreatic cell growth. In summary, SOCS3 inactivation by methylation was demonstrated to act in synergy with Reg3A overexpression to promote PaC cell growth and maybe the progress of inflammation-linked pancreatic carcinogenesis. KEY MESSAGES: Reg3A overexpression promoted cell growth in pancreatic cancer. SOCS3 is a key target in cancer by inhibiting cell growth and inducing apoptosis. SOCS3 negatively regulated Reg3A-mediated cell growth in pancreatic cancer. SOCS3 methylation act in synergy with Reg3A overexpression to promote pancreatic cancer cell growth.

[Preparation technology of total coumarins from Laportea bulbifera].

OBJECTIVE: To investigate the preparation process of total coumarins from Laportea bulbifera. METHODS: The optimum condition for the extraction of total coumarins was studied using the orthogonal test, and the extracts was purled with different kinds of macroporous resins. The content of total coumarins was detected by ultraviolet spectrophotometry. RESULTS: The optimum extraction process was as follows: three-time circumfluence with 4 times volume of 75% ethanol for 1.5 h each time. HPD300 macroporous resin was the optimum one for the separation and purification of total coumarins. The extraction condition was loading 30 mL of 0.4 g/mL the crude drug, flowing at 2BV/h, then edulcorating with 4 times volume of distilled water and eluting with the same volume of 75% ethanol. The yield rate was more than 52%. CONCLUSION: This method is suitable for the preparation of total coumarins in Laportea bulbifera, and the process is stable and repeatable.