Chunyou Wang

Establishment of clonal colony-forming assay for propagation of pancreatic cancer cells with stem cell properties.

Objective: Pancreatic cancer is among the most aggressive solid malignancies. It is possible that pancreatic cancer contains cancer stem cells responsible for its malignancy. The purposes of this study were (1) to establish an assay in which a subset of pancreatic cancer cell line (PANC-1) cells with stem cell properties can propagate, and (2) to identify the cells obtained from this assay. Methods: The PANC-1 cells were cultured in Dulbecco modified eagle medium F12 supplemented with epidermal growth factor, basic fibroblast growth factor, insulin, transferrin, selenium, and bovine serum albumin at a density of 1000 cells/mL for 10 to 14 days to form spheres. Cells of spheres were cultured in different conditions to evaluate their ability of self-renewal and differentiation. Clone formation assay and tumor formation assay were used to identify the ability of propagation in vitro and in vivo. The cells and spheres were also stained by using Hoechst 33342 dye to evaluate their capacity of excluding Hoechst dye. Real-time polymerase chain reaction was used to detect expressions of LY6E, c-Met, TACSTD1, CD34, and CD44 mRNA. Results: A subpopulation of PANC-1 cells could propagate to form spheres in this assay. Cells of obtained spheres had the hallmark of excluding Hoechst 33342 dye. Cultured in serum-free medium, the dissociated single cells of primary spheres could form filial spheres again; cultured in serum-containing medium, these cells generated both the cells with the ability of excluding Hoechst 33342 dye and the cells without that ability. The propagation capacity of PANC-1 spheres was higher than that of cells cultured in serum-containing medium both in vitro and in vivo. In addition, LY6E, TACSTD1, and CD44 mRNA were overexpressed in PANC-1 spheres. Conclusions: A subpopulation of PANC-1 cells can propagate to form spheres with properties of stem cells in this assay; enough of these cells can be obtained for further study. Considering the overexpression of mRNA, it was tentatively suggested that LY6E, TACSTD1, and CD44 proteins may act as surface markers for sorting pancreatic cancer stem cells with fluorescence-activated cell sorter/magnetic-activated cell sorter.

MiR-130b Is a Prognostic Marker and Inhibits Cell Proliferation and Invasion in Pancreatic Cancer through Targeting STAT3

Accumulating evidence indicates that microRNAs (miRNAs) are aberrantly expressed in human cancer and contribute to the tumorigenesis, but their roles in pancreatic cancer are still largely unknown. In this study, our data showed that miR-130b was significantly downregulated in 52 pairs of pancreatic cancer tissues and five cell lines. Furthermore, the deregulated miR-130b was correlated with worse prognosis, increased tumor size, late TNM stage, lymphatic invasion and distant metastasis. Multivariate analysis showed that miR-130b expression was a significant and independent prognostic predictor for pancreatic cancer patients. Functional studies indicated that the overexpression of miR-130b dramatically suppressed the proliferation of pancreatic cancer cells both in vitro and in vivo, which could be attributed to the induction of apoptosis and cell cycle arrest at S phase. Meanwhile, an overexpressed miR-130b remarkably inhibited the invasive ability of pancreatic cancer cells. Moreover, the dual luciferase assay revealed that STAT3 was directly targeted by miR-130b, which was further confirmed by the inverse expression of miR-130b and STAT3 in pancreatic cancer samples. Our findings suggested that miR-130b might have a considerable potential in prognosis identification and application of therapy for pancreatic cancer.

Superior removal of arsenic from water with zirconium metal-organic framework UiO-66

In this study, water stable zirconium metal-organic framework (UiO-66) has been synthesized and for the first time applied as an adsorbent to remove aquatic arsenic contamination. The as-synthesized UiO-66 adsorbent functions excellently across a broad pH range of 1 to 10, and achieves a remarkable arsenate uptake capacity of 303 mg/g at the optimal pH, i.e., pH = 2. To the best of our knowledge, this is the highest arsenate As(V) adsorption capacity ever reported, much higher than that of currently available adsorbents (5–280 mg/g, generally less than 100 mg/g). The superior arsenic uptake performance of UiO-66 adsorbent could be attributed to the highly porous crystalline structure containing zirconium oxide clusters, which provides a large contact area and plenty of active sites in unit space. Two binding sites within the adsorbent framework are proposed for arsenic species, i.e., hydroxyl group and benzenedicarboxylate ligand. At equilibrium, seven equivalent arsenic species can be captured by one Zr6 cluster through the formation of Zr-O-As coordination bonds.

miRNA-141, Downregulated in Pancreatic Cancer, Inhibits Cell Proliferation and Invasion by Directly Targeting MAP4K4

miRNAs are associated with various types of cancer due to their ability to affect expression of genes that modulate tumorigenesis. In this study, we explored the role of miR-141 in pancreatic cancer. The analysis of clinical characteristics showed that miR-141 was significantly downregulated in tissues and cell lines of pancreatic cancer. Moreover, the decreased miR-141 level was significantly associated with tumor size and TNM stage, as well as lymph node and distant metastasis. Meanwhile, both Kaplan–Meier and multivariate survival analysis showed decreased miR-141 were associated with overall survival. Overexpression of miR-141 in pancreatic cancer cells inhibited cell proliferation, clonogenicity, and invasion; induced G1 arrest and apoptosis; and enhanced chemosensitivity. To understand how miR-141 mediates the phenotype of pancreatic cancer cells, a bioinformatics tool was used to identify MAP4K4 as a potential target of miR-141. The Dual-Luciferase reporter gene assay showed that miR-141 binds directly to the 3′-untranslated region (3′UTR) of MAP4K4 to inhibit MAP4K4 expression. Western blot and quantitative real-time PCR (qRT-PCR) analyses revealed that MAP4K4 expression was inversely correlated with miR-141 expression both in pancreatic cancer samples and cell lines. Knockdown of MAP4K4 inhibited cell proliferation, clonogenicity, and invasion, induced G1 arrest and apoptosis, and enhanced chemosensitivity. In a nude mouse xenograft model, both overexpression of miR-141 and knockdown of MAP4K4 significantly repressed pancreatic cancer cell growth. Therefore, we conclude that miR-141 targets MAP4K4, acts as a tumor suppressor in pancreatic cancer cells, and may serve as a novel therapeutic agent for miRNA-based pancreatic cancer therapy. Mol Cancer Ther; 12(11); 2569–80. ©2013 AACR.

MiR-148b functions as a tumor suppressor in pancreatic cancer by targeting AMPKα1

miRNAs are small noncoding RNAs that participate in a variety of biologic processes, and dysregulation of miRNA is always associated with cancer development and progression. Aberrant expression of miR-148b has been found in some types of cancer, but its expression and potential biologic role in pancreatic cancer are still largely unknown. In this study, our data showed that miR-148b was significantly downregulated in 48 pairs of human pancreatic cancer tissues and five cell lines. Furthermore, the deregulated miR-148b was correlated with increased tumor size, late tumor–node–metastasis stage, lymphatic invasion, distant metastasis, and worse prognosis in pancreatic cancer. Functional studies indicated overexpression of miR-148b dramatically suppressed the growth of cancer cells, attributable to induction of apoptosis and cell-cycle arrest at S-phase. Meanwhile, miR-148b remarkably inhibited invasion and enhanced chemosensitivity of pancreatic cancer cells. Moreover, ectopic expression of miR-148b was able to inhibit tumorigenicity in nude mice. Further studies revealed that AMPKα1 might be the direct target gene of miR-148b, and overexpressed AMPKα1 inversely correlated with miR-148b in pancreatic cancer. Silencing of AMPKα1 with RNA interference inhibited the growth of pancreatic cancer cells in vitro and in vivo and also induced apoptosis, cell-cycle arrest, and inhibited invasion of cancer cells, which is consistent with the effects of miR-148b overexpression. In conclusion, miR-148b can inhibit cell proliferation, invasion, and enhance chemosensitivity of pancreatic cancer by targeting AMPKα1. Our present results implicate the potential effects of miR-148b on prognosis and treatment of pancreatic cancer. Mol Cancer Ther; 12(1); 83–93. ©2012 AACR.

Chronic pancreatitis and pancreatic cancer demonstrate active epithelial–mesenchymal transition profile, regulated by miR-217-SIRT1 pathway

Epithelial-mesenchymal transition (EMT) is supposed to be an inflammation induced response which may take a central role in tumorigenesis. Since recent evidence indicates that microRNAs may be involved in EMT, the present study set out to reveal the miRNA which might regulate the EMT in CP (chronic pancreatitis) and PC (pancreatic cancer) and its potential mechanism. Firstly, we provided evidence that both CP and PC tissues demonstrated active EMT profile. Consistently, miR-217 was obviously down-regulated in CP, PC and TGF-β1 treated PC cells, while negatively correlated to its direct target SIRT1. Moreover, either ectopic expression of miR-217 or inhibition of SIRT1 remarkably induced mesenchymal to epithelial transition (MET) in TGF-β1 treated PC cells. On the contrary, miR-217 inhibitor promoted EMT in PC cells but not in SIRT-knockdown PC cells. Clinical information from a cohort of 54 PC patients demonstrated that down-regulated miR-217 was positively correlated with late tumor stage, lymphatic invasion, vascular infiltration and distant metastasis. These results suggest that the overexpressed TGF-β1 in inflammation triggers the deregulation of the miR-217-SIRT1 pathway and then promotes the EMT process, which might be involved in the tumorigenesis of PC. Additionally, miR-217 may function as a novel target and predictor for PC prevention and therapy.

Blockade of sonic hedgehog signal pathway enhances antiproliferative effect of EGFR inhibitor in pancreatic cancer cells

AbstractAim:To investigate the expression of sonic hedgehog (SHH) and epidermal growth factor receptor (EGFR) signal molecules in pancreatic cancer cells, and to assess the inhibitory effects through the blockade of the SHH and EGFR signaling pathways by cyclopamine and Iressa, respectively.Methods:The expression of SHH and EGFR in pancreatic cancer cell lines (PANC-1, SUIT-2, and ASPC-1) was detected by RT-PCR and Western blot analysis. After treatment with different concentrations of cyclopamine, alone or in combination with Iressa, the antiproliferative effect on pancreatic cancer cells was analyzed by methyl thiazolyl tetrazolium assays. A flow cytometry analysis was used to detect the cellular cycle distribution and apoptosis of pancreatic cancer cells.Results:All of the 3 pancreatic cancer cell lines expressed SHH, Smoothened (SMO), and EGFR. Cyclopamine could downregulate the expression of EGFR in all cell lines. Cyclopamine or Iressa could induce a growth inhibitory effect in a dose-dependent manner. Moreover, the combined use of 2.5 μmol/L cyclopamine and 1 μmol/L Iressa induced an enhanced inhibitory effect and a greater apoptosis rate than any agent alone. The percentage of the cell population of the G0/G1 and sub-G1 phases was significantly increased along with the increasing dose of cyclopamine and/or Iressa.Conclusion:The blockade of the sonic hedgehog signal pathway enhances the antiproliferative effect of the EGFR inhibitor through the downregulation of its expression in pancreatic cancer cells. The simultaneous blockade of SHH and EGFR signaling represents possible targets of new treatment strategies for pancreatic carcinoma.

Mammalian ncRNA-disease repository: a global view of ncRNA-mediated disease network

Dear Editor  Recently, substantial studies have begun to explore the functional diversity and mechanistic roles of ncRNAs in mammals.1 Now, it has become increasingly apparent that ncRNAs are involved in multiple major biological processes, such as developmental timing, fat metabolism and cell death.2 Furthermore, the epigenetic and genetic defects in ncRNAs and their processing machinery have been implicated in the etiology of many forms of diseases.3 Several databases that documented the relevance of the microRNAs(miRNAs) to diseases have been constructed and provided useful results.4, 5 However, miRNAs are just the tip of the iceberg, other ncRNAs such as long non-coding RNAs (lncRNAs), PIWI-interacting RNAs (piRNAs) and small nucleolar RNAs (snoRNAs) have also been demonstrated to contribute to diseases.3, 6 Accumulated evidence suggest the diverse non-coding RNAs (ncRNAs) involved in a wide variety of diseases progression.3, 6, 7 It is a key challenge for understanding the precise behavior of diverse ncRNAs in mammalian diseases and deciphering the cross-regulations among disease-associated ncRNAs. Because there was no repository focused on diverse ncRNA-disease relationships in mammals, we have developed a manually curated diverse ncRNA-disease repository (MNDR, www.rna-society.org/mndr/) by integrating evidence in three mammals. Totally, 807 lncRNA-associated, 229 miRNA-associated, 13 piRNA-associated and 100 snoRNA-associated entries for 1149 curated entries were documented for three mammals (866 Homo sapiens-associated, 251 Mus musculus-associated and 32 Rattus norvegicus-associated entries) (Table 1). Table 1 The statistics of the ncRNA-disease entries in MNDR database Recent investigations indicated there are complex regulations among diverse ncRNAs and protein-coding genes. Such as, PTEN gene and the PTEN pseudogenes (ptenp1, one of lncRNAs) share a high degree of sequence homology, changes in ptenp1 expression levels indirectly affect PTEN expression by sequestering PTEN-targeting miRNAs.8 Thus understanding the mutual regulating pattern among diverse ncRNAs and protein-coding genes, particularly in disease conditions, is a key challenge. Thus, MNDR is not only a knowledge depository but providing us a good opportunity to view the ncRNA-mediated disease network globally (in visualization page: www.rna-society.org/mndr/visualization.html). Diverse ncRNAs and interaction genes were represented as nodes and the regulations were denoted as edges. Based on such a simplified ncRNA-mediated disease network, interesting observations have been achieved. The result showed that snoRNA htr, as a hub node, has intensively linked to 21 interaction genes in the network. More important, through BCL2, BCL2L1 and BAX, the snoRNA htr can communicate with the lncRNA malat1 (Supplementary Figure 1). Another example is snoRNA htr and lncRNA h19 are linked by E2F1 and MYC. When combined with human disease-associated miRNA evidence from mir2disease database, lncRNAs, miRNAs and snoRNAs, together with their interaction/target genes, can be integrated into bigger expanding ncRNA-mediated disease network. The biggest sub-network has 129 nodes and 149 edges, involving 33 lncRNAs, 1 snoRNA, 19 miRNAs and 76 interaction protein-coding genes(Supplementary Figure 2). In this network, more regulations among diverse ncRNAs directly or indirectly via intermediate genes, lncRNA dgcr5, har1a and har1b, were connected with hsa-mir-21 via intermediate gene REST. Interestingly, hsa-mir-21 and snoRNA htr were linked by key anti-apoptosis gene BCL2. Similar results were observed that lncRNA dgcr5, har1a and har1b can also communicate with snoRNA htr through alternative route NFKB1-hsa-mir-9-REST. Hence, according to current data, the two pivot protein-coding genes (BCL2 and NFKB1) and several ncRNAs (lncRNA malat1, snoRNA htr and miRNA hsa-mir-21, has-mir-9) collectively play an important role in the ncRNA-mediated disease network (Supplementary Figure 2). Importantly, the crosstalk between lncRNA malat1 and miRNA hsa-mir-21 can be found conserved in mouse ncRNA-mediated disease network. Above observations indicated diverse ncRNAs could communicate with each other in disease state through some disease-associated genes in mammals, highlighting the complexity, conservative and plasticity of the regulatory relationships between diverse ncRNAs and protein-coding genes in diseases.

Cancer Stem-Like Cells Enriched in Panc-1 Spheres Possess Increased Migration Ability and Resistance to Gemcitabine

Pancreatic cancer is one of the most lethal malignancies with poor prognosis. Previously, we found that a subpopulation of cancer stem cells (CSCs) in the Panc-1 pancreatic cancer cell line could propagate to form spheres. Here we characterized the malignant phenotypes of the pancreatic cancer stem CD44+/CD24+ cells, which were enriched under sphere forming conditions as analyzed by flow cytometry. These cells demonstrated increased resistance to gemcitabine and increased migration ability. Moreover, these cells exhibited epithelial to mesenchymal transition characterized by a decreased level of the epithelial marker E-cadherin and an increased level of the mesenchymal marker vimentin. Notably, abnormal expression of Bmi-1, ABCG2, Cyclin D1 and p16 were found in Panc-1 CSCs. Our results suggest that targeted inhibition of CSCs represents a novel therapeutic approach to overcome chemoresistance and metastasis of pancreatic cancer.

Effects of different resuscitation fluid on severe acute pancreatitis

AIM To compare effects of different resuscitation fluid on microcirculation, inflammation, intestinal barrier and clinical results in severe acute pancreatitis (SAP). METHODS One hundred and twenty patients with SAP were enrolled at the Pancreatic Disease Institute between January 2007 and March 2010. The patients were randomly treated with normal saline (NS group), combination of normal saline and hydroxyethyl starch (HES) (SH group), combination of normal saline, hydroxyethyl starch and glutamine (SHG group) in resuscitation. The ratio of normal saline to HES in the SH and SHG groups was 3:1. The glutamine (20% glutamine dipeptide, 100 mL/d) was supplemented into the resuscitation liquid in the SHG group. Complications and outcomes including respiratory and abdominal infection, sepsis, abdominal hemorrhage, intra-abdominal hypertension, abdominal compartment syndrome (ACS), renal failure, acute respiratory distress syndrome (ARDS), multiple organ dysfunction syndrome (MODS), operation intervention, length of intensive care unit stay, length of hospital stay, and mortality at 60 d were compared. Moreover, blood oxygen saturation (SpO2), gastric intramucosal pH value (pHi), intra-abdominal pressure (IAP), inflammation cytokines, urine lactulose/mannitol (L/M) ratio, and serum endotoxin were investigated to evaluate the inflammatory reaction and gut barrier. RESULTS Compared to the NS group, patients in the SH and SHG groups accessed the endpoint more quickly (3.9 ± 0.23 d and 4.1 ± 0.21 d vs 5.8 ± 0.25 d, P < 0.05) with less fluid volume (67.26 ± 28.53 mL/kg/d, 61.79 ± 27.61 mL/kg per day vs 85.23 ± 21.27 mL/kg per day, P < 0.05). Compared to the NS group, incidence of renal dysfunction, ARDS, MODS and ACS in the SH and SHG groups was obviously lower. Furthermore, incidence of respiratory and abdominal infection was significantly decreased in the SH and SHG groups, while no significant difference in sepsis was seen. Moreover, less operation time was needed in the SH and SHG group than the NS group, but the difference was not significant. The mortality did not differ significantly among these groups. Blood SpO2 and gastric mucosal pHi in the SH and SHG groups increased more quickly than in the NS group, while IAP was significantly decreased in the SH and SHG group. Moreover, the serum tumor necrosis factor-α, interleukin-8 and C-reactive protein levels in the SH and SHG groups were obviously lower than in the NS group at each time point. Furthermore, urine L/M ratio and serum endotoxin were significantly lower in the SH group and further decreased in the SHG group. CONCLUSION Results indicated that combination of normal saline, HES and glutamine are more efficient in resuscitation of SAP by relieving inflammation and sustaining the intestinal barrier.