Chuan-Yong Guo

Reg4 protects against acinar cell necrosis in experimental pancreatitis.

Background and aims Reg4 is a recently discovered member of the regenerating gene family with distinctive expression profiles in primary cancers. To date, the physiological function of Reg4 is poorly understood. Previously, the authors found that Reg4 was markedly upregulated during acute pancreatitis (AP). The aim of this study was to investigate the role of Reg4 in experimental pancreatitis. Methods AP was induced in C57BL/6 mice by administration of either l-arginine or caerulein, and Reg4 expression was assessed by immunofluorescence, reverse transcriptase (RT)-PCR and western blot analyses. Recombinant human Reg4 protein (rReg4), heat-inactivated Reg4, neutralising antibody and vehicle were also administered to mice by subcutaneous injection. The severity of AP was determined by measuring amylase and lipase activities in the serum and histological grading. The effect of rReg4 on cell death was examined and epidermal growth factor receptor (EGFR), p-EGFR, Akt, p-Akt, Bcl-2 and Bcl-xL expression were assessed by western blot analysis of isolated murine acinar cells treated with l-arginine. Results Reg4 mRNA and protein were markedly upregulated during arginine-induced pancreatitis. Reg4 was widely expressed in residual acinar cells around the islets and regenerating metaplastic epithelium. rReg4 could protect against arginine-induced necrosis of acinar cells both in vivo and in vitro. This protective effect was also confirmed in the caerulein-induced murine model of AP. It was shown that arginine induced expression of Bcl-2 and Bcl-xL, while rReg4 upregulated Bcl-2 and Bcl-xL expression by activating the EGFR/Akt pathway. The upregulation of Bcl-xL correlated inversely with cell necrosis in isolated pancreatic acinar cells. Conclusions The data suggest that Reg4 may protect against acinar cell necrosis in experimental pancreatitis by enhancing the expression of Bcl-2 and Bcl-xL via activation of the EGFR/Akt signalling pathway.

Expression of DNMT1 and DNMT3a Are Regulated by GLI1 in Human Pancreatic Cancer

Background and Aims GLI1, 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. Methods Expressions 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. Results IHC 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. Conclusion DNMT1 and DNMT3a are regulated by GLI1 in PC, and DNMT1 is its direct target gene.

Resveratrol Inhibits Proliferation and Induces Apoptosis through the Hedgehog Signaling Pathway in Pancreatic Cancer Cell

Purpose: To investigate the effect and possible mechanisms of resveratrol on pancreatic cancer cells in vitro. Methods: After being treated with resveratrol, cell viability, cell cycle phase distribution and apoptosis rate of pancreatic cancer cells were measured by CCK-8 assay and flow cytometer, respectively. The effects of resveratrol on the Hedgehog pathway were studied by real-time RT-PCR and Western blotting. By interfering Gli1 expression in PANC-1 cells and overexpressing Gli1 in BxPC-3 cells, we detected the expressions of Gli1-targeted genes, such as Ptc1, CCND1 and BCL-2, compared with resveratrol experimental group. We further used the luciferase reporter assay to explore the correlation between resveratrol and Gli1. Results: Resveratrol inhibited the growth of pancreatic cancer cells in a dose- and time-dependent manner. Compared with control group, the cells in the G0/G1 phase and the apoptosis rate were significantly increased. Low concentration of resveratrol decreased the expression of the Hedgehog pathway members including Gli1, Ptc1 and Smo. The expression of downstream target genes of the Hedgehog pathway such as Gli1, Ptc1, CCND1 and BCL-2 were significantly decreased after 12.5 µM resveratrol treatment, which demonstrated a similar change of gene expression when Gli1 was knocked down by the RNAi technique in PANC-1 cells. Resveratrol also downregulated the expression of Gli1, Ptc1, CCND1 and BCL-2 in Gli1-overexpressed BxPC-3 cells. Results of the luciferase assay showed that resveratrol did not act on the Gli1 promoter directly. Conclusion: Resveratrol can inhibit pancreatic cancer cell survival and its mechanisms might be partly via the Hedgehog signaling pathway.

Meta-analysis of the efficacy of probiotics in Helicobacter pylori eradication therapy

AIMnTo evaluate the role of probiotics in the standard triple Helicobacter pylori therapy.nnnMETHODSnIn this meta-analysis, we investigated the efficacy of probiotics in a standard triple H. pylori therapy in adults. Searches were mainly conducted in MEDLINE/PubMed, EMBASE, and the Cochrane Central Register of Controlled Trials. Fourteen studies met our criteria, and the quality of these studies was assessed using the Jadad scale. We used STATA version 12.0 to extract data and to calculate the odds ratios (ORs), which are presented with the corresponding 95% confidence intervals (CIs). The data are presented as forest plots.nnnRESULTSnThe pooled ORs for the eradication rates calculated by intention-to-treat analysis and per-protocol analysis in the probiotic group vs the control group were 1.67 (95%CI: 1.38-2.02) and 1.68 (95%CI: 1.35-2.08), respectively, using the fixed-effects model. The sensitivity of the Asian studies was greater than that of the Caucasian studies (Asian: OR = 1.78, 95%CI: 1.40-2.26; Caucasian: OR = 1.48, 95%CI: 1.06-2.05). The pooled OR for the incidence of total adverse effects was significantly lower in the probiotic group (OR = 0.49, 95%CI: 0.26-0.94), using the random effects model, with significant heterogeneity (I (2) = 85.7%). The incidence of diarrhea was significantly reduced in the probiotic group (OR = 0.21, 95%CI: 0.06-0.74), whereas the incidence of taste disorders, metallic taste, vomiting, nausea, and epigastric pain did not differ significantly between the probiotic group and the control group.nnnCONCLUSIONnSupplementary probiotic preparations during standard triple H. pylori therapy may improve the eradication rate, particularly in Asian patients, and the incidence of total adverse effects.

Down-regulation of miR-212 expression by DNA hypermethylation in human gastric cancer cells

There has been few report discussing the expression and function of miR-212 in gastric cancer (GC). The aim of this pilot study was to investigate the expression of miR-212 in both gastric cancer tissues and gastric cancer cells and further explores the possible reasons for this change and the impact on the development of gastric cancer. qRT–PCR was used to detect the expression of miR-212 in primary GC tissues, adjacent normal tissues, gastric cancer cell lines BGC-823, SGC-7901, MKN-45, and normal gastric mucosa cell line GES. The expression of miR-212 was evaluated before and after treatment with methylation inhibitor-5-Aza-2′-deoxycitidine (5-Aza-dC), finally anti-miRNA and dual luciferase reporter assay were used to prove that MYC is a target gene of miR-212. The results showed that a significant reduction of miR-212 expression in GC tissues was observed compared to that in normal tissues (Pxa0=xa00.002). At the same time, miR-212 expression level in normal gastric mucosa cell line GES was higher than that of in gastric cancer cell lines BGC-823, SGC-7901, and MKN-45 (Pxa0=xa00.015, 0.008, 0.044, respectively). Computer sequence analysis showed the hypermethylation of CpG islands(CPI) in the promoter regions of miR-212 led to the lower expression of miR-212 in gastric cell strains (BGC-823 and SGC-7901). MiR-212 expression was significantly recovered after treatment with methylation inhibitor 5-Aza-dC (Pxa0=xa00.016, 0.000, 0.015, respectively). Then, the results of AMOs transfection and dual luciferase reporter assay showed that Myc is a target of miR-212, which will be helpful to verify the function of miR-212 in carcinogenesis. The conclusion could be deduced from the study that decreased expression of miR-212 may be due to hypermethylation of CPI in gastric cancer cells, and miR-212 might act on the progression of gastric cancer through the potential target gene Myc.

Gli1 maintains cell survival by up-regulating IGFBP6 and Bcl-2 through promoter regions in parallel manner in pancreatic cancer cells

Background: Aberrant activation of Hedgehog (Hh) signaling pathway has been reported to be related to malignant biological behavior of pancreatic cancer but its mechanism is unclear yet. Since IGF pathway and Bcl-2 family are involved in proliferation and apoptosis of pancreatic cancer cells, we hypothesize that they are possibly associated with Hh pathway. Materials and Methods: We studied the relationship of Shh-Gli1 signaling pathway with proliferation and apoptosis of pancreatic cancer cells and the regulation of transcription factor Gli1 to insulin-like growth factor binding protein 6 (IGFBP6) and Bcl-2 genes at the level of transcription. Results: Sonic hedgehog (Shh), Smoothened (Smo), patched and Gli1 were expressed in pancreatic cancer cells. Cyclopamine inhibited cell proliferation at low concentration and induced apoptosis at high concentration. Effect of RNA interference (RNAi) for Gli1 to cell survival is mainly due to proliferation inhibition though involved in apoptosis. The transcription factor Gli1 bound to promoter regions of Bcl-2 and IGFBP6 genes and the levels of IGFBP6, proliferating cell nuclear antigen (PCNA) and Bcl-2 messenger RNA (mRNA) were decreased as well as Gli1 mRNA significantly by cyclopamine or RNAi in cultured pancreatic cancer cells (p < 0.01). Finally PCNA, IGFBP6 and Bcl-2 mRNA were upregulated as well as Shh or Gli1 in pancreatic cancer tissues (p < 0.01). Conclusions: Our study reveals that Gli1 maintained cell survival by binding the promoter regions and facilitating transcription of IGFBP6 and Bcl-2 genes in a parallel manner in pancreatic cancer cells and suggests it may be one of the mechanisms of Shh-Gli1 signaling pathway in pancreatic cancer.

Identification of RegIV as a novel GLI1 target gene in human pancreatic cancer.

Background and Aims GLI1 is the key transcriptional factor in the Hedgehog signaling pathway in pancreatic cancer. RegIV is associated with regeneration, and cell growth, survival, adhesion and resistance to apoptosis. We aimed to study RegIV expression in pancreatic cancer and its relationship to GLI1. Methods GLI1 and RegIV expression were evaluated in tumor tissue and adjacent normal tissues of pancreatic cancer patients and 5 pancreatic cancer cell lines by qRT-PCR, Western blot, and immunohistochemistry (IHC), and the correlation between them. The GLI1-shRNA lentiviral vector was constructed and transfected into PANC-1, and lentiviral vector containing the GLI1 expression sequence was constructed and transfected into BxPC-3. GLI1 and RegIV expression were evaluated by qRT-PCR and Western blot. Finally we demonstrated RegIV to be the target of GLI1 by chromatin immunoprecipitation (CHIP) and electrophoretic mobility shift assays (EMSA). Results The results of IHC and qRT-PCR showed that RegIV and GLI1 expression was higher in pancreatic cancer tissues versus adjacent normal tissues (p<0.001). RegIV expression correlated with GLI1 expression in these tissues (Ru200a=u200a0.795, p<0.0001). These results were verified for protein (Ru200a=u200a0.939, pu200a=u200a0.018) and mRNA expression (Ru200a=u200a0.959, pu200a=u200a0.011) in 5 pancreatic cancer cell lines. RegIV mRNA and protein expression was decreased (94.7±0.3%, 84.1±0.5%; respectively) when GLI1 was knocked down (82.1±3.2%, 76.7±2.2%; respectively) by the RNAi technique. GLI1 overexpression in mRNA and protein level (924.5±5.3%, 362.1±3.5%; respectively) induced RegIV overexpression (729.1±4.3%, 339.0±3.7%; respectively). Moreover, CHIP and EMSA assays showed GLI1 protein bound to RegIV promotor regions (GATCATCCA) in pancreatic cancer cells. Conclusion GLI1 promotes RegIV transcription by binding to the RegIV gene promoter in pancreatic cancer.

Anticancer Effect of Celecoxib via COX-2 Dependent and Independent Mechanisms in Human Gastric Cancers Cells

Cyclooxygenase-2 (COX-2) inhibitors cause growth inhibition of human gastric carcinoma cells, but it remains unclear whether this is both COX-2 dependent and independent. The related mechanisms remain to be determined. Both low COX-2 expressing gastric carcinoma and high COX-2 expressing gastric carcinoma cells were used to study the effect and mechanisms of celecoxib on gastric carcinoma cell growth. Celecoxib resulted in comparable growth inhibition in AGS cells with stable transfections of small interfering RNA (siRNA) against COX-2 (SAC) and negative control vector (NC) cells. Simultaneously, celecoxib resulted in significant reduction of Bcl-2 and significant increase of p21WAF1 and p27KIP1 in SAC and NC cells. The present study shows that celecoxib causes growth inhibition of gastric carcinoma cells by decreasing Bcl-2 of cyclooxygenase-2-dependent pathway, and by increasing p21WAF1 and p27KIP1 of cyclooxygenase-2-independent pathway. These data extend our knowledge on the effect and mechanisms of celecoxib-induced inhibition of gastric carcinoma cell growth.

Increased expression of cellular repressor of E1A-stimulated gene (CREG) in gastric cancer patients: a mechanism of proliferation and metastasis in cancer.

BackgroundThe cellular repressor of E1A-stimulated genes (CREG), a secreted glycoprotein, has been studied with human embryonic carcinoma cells, vascular smooth muscle cells, and NIH3T3 fibroblasts. However, its relationship to tumor cell proliferation and metastasis has not been examined in human gastric cancers (GC) until now.AimTo investigate the expression of CREG in GC and its association with GC cell proliferation and metastasis.MethodsForty-two cases of GCs, matched normal gastric tissues, and the human gastric cancer cell lines BGC-823, SGC-7901, MKN45, normal gastric mucosa cell line GES, and HUVEC cell line ECV304 were used to analyze CREG expression at the level of mRNA and protein. The expression of CREG was then further examined by immunohistochemistry in 42 GC tissues, and the correlation between the level of CREG and the pathological and clinical data was evaluated. Finally, we down-regulated the expression of CREG in GC cells with specific siRNA, and assessed the role of CREG in the proliferation and metastasis/invasion of the GC cell line.ResultsThe level of CREG was found to be higher in malignant GC tissues and cells compared to adjacent normal tissues and normal gastric cells (pxa0<xa00.001). Additionally, the expression levels of CREG were positively correlated with tumor clinical stage (pxa0=xa00.001), tumor metastasis (pxa0<xa00.001), and stages of tumor infiltration (pxa0=xa00.019). Furthermore, by using siRNA, we found that the down-regulated expression of CREG inhibited the proliferation of GC cells (pxa0<xa00.05), and migration of both GC cells (pxa0=xa00.001).ConclusionsOur data suggest that CREG plays an important role in gastric cancer cell proliferation and metastasis and that CREG may be a potential therapeutic target for GC.

Efficacy of intraductal ultrasonography in the diagnosis of non-opaque choledocholith

AIMnTo evaluate the efficacy of intraductal ultrasonography (IDUS) in the diagnosis of non-opaque, common bile duct stones.nnnMETHODSnA total of 183 patients (102 males, mean age 73 years; 81 females, mean age 70 years) with suspected common bile duct stones diagnosed through abdominal computed tomography (CT), magnetic resonance imaging (MRI), and abdominal Type-B ultrasound were included in the study. The diagnosis was confirmed through endoscopic retrograde cholangiopancreatography (ERCP) followed by IDUS.nnnRESULTSnA total of 183 patients with suspected common bile duct (CBD) stones were included in the study as follows: 36 patients with high-density CBD stones, 68 patients with sand-like stones, 44 patients with low-density stones, 21 patients with ampullary cancer, and 14 patients with pancreatic cancer. Conventional imaging revealed 124 cases of choledochectasia, and only 36 cases of suspected CBD stones; ERCP revealed 145 cases of CBD stones with three missed diagnoses. IDUS revealed 148 cases of CBD stones, 21 cases of ampullary tumors, and 14 cases of pancreatic cancer.nnnCONCLUSIONnIDUS was more effective in the diagnosis of bile duct stones than ERCP, upper abdominal CT or upper abdominal MRI.