Xiong Chen

miR-142-5p promotes development of colorectal cancer through targeting SDHB and facilitating generation of aerobic glycolysis

Aberrant expression of miRNAs contributes to the development of human malignancies. A recent study revealed that miR-142-5p is increased in the serum of colorectal cancer (CRC) patients compared to health people. Using starBase v2.0, we found that succinate dehydrogenase-B (SDHB) is a potential target of miR-142-5p, while SDHB is negatively correlated to cancer development through regulating energetic metabolism. Based on these information, this study further examined the expression profiles of miR-142-5p and SDHB in CRC tissues and cell lines using PCR and Western blotting. Transfection experiment and luciferase assay were performed to identify relationship between miR-142-5p and SDHB. Oxygen intake, glucose consumption and production of lactic acid were used to evaluate the influence on energetic metabolism. CRC growth and proliferation were assessed by in vitro and in vivo studies. Results showed that miR-142-5p was up-regulated in CRC, but SDHB was down-regulated. SDHB was confirmed as a target of miR-142-5p, and decreased SDHB in CRC was result from the abnormal up-regulation of miR-142-5p. Lose of SDHB by miR-142-5p inhibited oxygen intake by CRC cells, but increased glucose consumption and lactate production. These suggest miR-142-5p up-regulation in CRC probably facilitates generation of aerobic glycolysis by reducing SDHB. miR-142-5p promoted proliferation and colony formation of CRC, but inhibited apoptosis. SDHB overexpression abrogated these effect of miR-142-5p, which indicates that SDHB depletion mediates tumor-promoting actions of miR-142-5p. This study added novel insight into the CRC development regulated by miR-142-5p. It may be a promising therapy target in the future molecular therapy.

Stepwise sedation for elderly patients with mild/moderate COPD during upper gastrointestinal endoscopy

AIMnTo investigate stepwise sedation for elderly patients with mild/moderate chronic obstructive pulmonary disease (COPD) during upper gastrointestinal (GI) endoscopy.nnnMETHODSnEighty-six elderly patients with mild/moderate COPD and 82 elderly patients without COPD scheduled for upper GI endoscopy were randomly assigned to receive one of the following two sedation methods: stepwise sedation involving three-stage administration of propofol combined with midazolam [COPD with stepwise sedation (group Cs), and non-COPD with stepwise sedation (group Ns)] or continuous sedation involving continuous administration of propofol combined with midazolam [COPD with continuous sedation (group Cc), and non-COPD with continuous sedation (group Nc)]. Saturation of peripheral oxygen (SpO2), blood pressure, and pulse rate were monitored, and patient discomfort, adverse events, drugs dosage, and recovery time were recorded.nnnRESULTSnAll endoscopies were completed successfully. The occurrences of hypoxemia in groups Cs, Cc, Ns, and Nc were 4 (9.3%), 12 (27.9%), 3 (7.3%), and 5 (12.2%), respectively. The occurrence of hypoxemia in group Cs was significantly lower than that in group Cc (P < 0.05). The average decreases in value of SpO2, systolic blood pressure, and diastolic blood pressure in group Cs were significantly lower than those in group Cc. Additionally, propofol dosage and overall rate of adverse events in group Cs were lower than those in group Cc. Finally, the recovery time in group Cs was significantly shorter than that in group Cc, and that in group Ns was significantly shorter than that in group Nc (P < 0.001).nnnCONCLUSIONnThe stepwise sedation method is effective and safer than the continuous sedation method for elderly patients with mild/moderate COPD during upper GI endoscopy.

Effects of different types of Helicobacter pylori on the gap junction intercellular communication in GES-1 cells

OBJECTIVEnTo determine the effect of different types of Helicobacter pylori (H.pylori) on the gap junction intercellular communication (GJIC) in GES-1 cells, and investigate the types of H.pylori related to the dysfunction of GJIC.nnnMETHODSnDifferent types of H.pylori clinical strains were isolated and cultured, including the East Asian CagA(-)positive H.pylori (East Asian CagA(+)H.pylori), Western CagA(-)positive H.pylori (Western CagA(+)H.pylori), and the CagA(-)negative H.pylori (CagA(-)H.pylori). We co-cultured these H.pylori strains with GES-1 cells for 24 and 48 h, respectively. The control group was cultured without any H.pylori for 24 and 48 h. Change of the GJIC function in GES-1 cells was detected by the scrape-loading dye transfer (SLDT) technique. The cell proliferation of each group was examined by the methyl thiazolyl tetrazolium bromide (MTT) assay.nnnRESULTSnThe control group showed better GJIC function in the GES-1 cells, and the fluorescent dye migrated 4-5 rows to the adjacent cells at 24 and 48 h. Compared with the control group, the GJIC function of GES-1 cells in the CagA(-)H.pylori group decreased and the fluorescent dye migrated 3 rows to the adjacent cells. Compared with the control group and the CagA(-) H.pylori group, the GJIC function of GES-1 cells in the Western CagA(+)H.pylori group decreased and the fluorescent dye migrated 1-2 rows to the adjacent cells. The East Asian CagA(+)H.pylori group showed no GJIC function or weak GJIC function, and most of the fluorescent dye was confined to the area of scratched single row cells and only a few migrated 1-2 rows to the adjacent cells. Difference in the cell proliferation between the CagA(-)H.pylori group and the control group was not significant. The cell proliferation of the Western CagA(+)H.pylori group and the East Asian CagA(+)H.pylori group at bacterium-to-cell ratio of 100:1 and 200:1 was higher than that of the control group. The cell prolife-ration of the East Asian CagA(+)H.pylori group at bacterium-to-cell ratio of 400:1 was significantly lower than that of the control group at 48 h.nnnCONCLUSIONnH.pylori can inhibit the GJIC function in GES-1 cells, which may be associated with CagA(+)H.pylori, especially with East Asian CagA(+)H.pylori. The effect of H.pylori on the proliferation of GES-1 cells is related to virulence factor CagA.

L-ascorbic Acid-2-Glucoside inhibits Helicobacter pylori-induced apoptosis through mitochondrial pathway in Gastric Epithelial cells

Helicobacter pylori (H. pylori) infection is the major cause for gastritis, peptic ulcer, and gastric cancer. Elevated oxidative stress, mitochondrial dysfunction and apoptotic death of gastric epithelial cells are typical hallmarks of H. pylori infection. Ascorbic Acid 2-Glucoside (AA2G) is a stable version of Vitamin C, that binds glucose to conventional vitamin C. AA2G has free radical scavenging activities and anti-apoptotic abilities. However, the protective effect of AA2G against H. pylori-infection in gastric epithelial cells is yet unknown. In this study, we investigated the effects of AA2G in human H. pylori-infected gastric epithelial cells. AA2G could remarkably ameliorate H. pylori-induced oxidative stress, including the levels of intracellular reactive oxygen species (ROS) and 4-hydroxynonenal (4-HNE). Importantly, AA2G treatment also improved mitochondrial function by restoring the level of ATP and mitochondrial membrane potential (MMP). Furthermore, AA2G reduced apoptosis induced by H. pylori through modulation of mitochondria-dependent apoptotic pathways. Our findings suggest that AA2G has a protective effect against H. pylori infection in gastric epithelial cells.

Correlation between the cystathionine-r-lyase (CES) and the severity of peptic ulcer disease

BACKGROUNDnThe infection of Helicobacter pylori (H. pylori) is one of the most important causes of gastric ulcer disease. The role of hydrogen sulfide (H2S) production in H. pylori-induced gastric ulcer disease.nnnAIMnThe expression of cystathionine-γ-lyase (CSE) was determined, and correlated with the severity of gastric ulcer disease.nnnMETHODSnOne hundred and eight patients were selected based on the determination of gastric ulcer and the infection of Helicobacter pylori (H. pylori), including 36 normal control, 36 patients with H. Pylori-negative gastric ulcer, and 36 patients with H. Pylori-positive gastric ulcer. RT-PCR determination was performed to determine the expression of CSE, NF-κB and IL-8.nnnRESULTSnThe expression of CSE, NF-κB and IL-8 was higher in the gastric ulcer group than control group (p<0.05). Compared with the H. pylori-negative gastric ulcer, the expression of CSE, NF-κB and IL-8 was higher than H. pylori-positive gastric ulcer group (p<0.05). For H. pylori-negative gastric ulcer group, the expression of CSE positively correlated with the expression of NF-κB (r=0.98, p<0.05) and IL-8 (r=0.95, p<0.05). For H. pylori-positive gastric ulcer group, the expression of CSE also positively correlated with the expression of NF-κB (r=0.99, p<0.05) and IL-8 (r=0.85, p<0.05).nnnCONCLUSIONnThe expression of CSE was positively correlated with the severity of gastric ulcer.

Inhibition of lncRNA NEAT1 suppresses the inflammatory response in IBD by modulating the intestinal epithelial barrier and by exosome-mediated polarization of macrophages

Inflammatory bowel disease (IBD) is a multifactorial inflammatory disease, and increasing evidence has demonstrated that the mechanism of the pathogenesis of IBD is associated with intestinal epithelial barrier injury. Long non‑coding RNAs (lncRNAs) are a class of transcripts >200 nucleotides in length with limited protein‑coding capability. Nuclear paraspeckle assembly transcript 1 (NEAT1) is a recently identified nuclear‑restricted lncRNA, which localizes in subnuclear structures, termed paraspeckles, and is involved in the immune response in a variety of ways. However, the function of NEAT1 in IBD remains to be fully elucidated. In the present study, reverse transcription‑quantitative polymerase chain reaction assays were performed to determine the expression levels of NEAT1 lncRNA in IBD serum samples and tissues. Furthermore, the effect of NEAT1 on the cell permeability of colon cells was investigated via determination of trans‑epithelial electrical resistance as well as performance of western blot and immunofluorescence assays. In addition, dextran sodium sulfate assays were performed to investigate the effect of downregulation of NEAT1 in IBD of mice. The present study detected the expression levels of NEAT1 in IBD cells and animal models to examine the changes in intestinal epithelial cell permeability following inhibition of the expression of NEAT1. In addition, phenotypic transformation was examined following different treatments in epithelial cells and macrophages. The results suggested that the expression of NEAT1 was high in IBD and was involved in the inflammatory response by regulating the intestinal epithelial barrier and through exosome‑mediated polarization of macrophages. The downregulation of NEAT1 suppressed the inflammatory response by modulating the intestinal epithelial barrier and through exosome‑mediated polarization of macrophages in IBD. The results of the present study revealed a potential strategy of targeting NEAT1 for IBD therapy.

Protective Role of Coxsackie-Adenovirus Receptor in the Pathogenesis of Inflammatory Bowel Diseases

Aim To investigate the role of Coxsackie-adenovirus receptor (CAR) in inflammatory bowel disease (IBD). Background CAR, a type I transmembrane protein with functions in virus attachment, has been shown to be associated with epithelial tight junctions (TJs) and mediates cell adhesion, implying its potential roles in the pathogenesis of IBD. Methods and Materials To determine the effect of CAR in IBD using QPCR and Western blotting to determine the expression of CAD in TNF-α induced NCM460 and SW480 cells and IBD tissues compared to control groups. Furthermore, TJs dysregulation, FITC-Dextran permeability assay, qRT-PCR, Western blot, and IF assessed the permeability in CAR overexpressed cells treated with TNF-α. HE, qRT-PCR, Western blot, and IHC assay were used to assess the CAR overexpressed cells whether they have the effect to cure DSS induced ulcerative colitis rat model in vivo. Result We found CAR levels in human colon cell lines are significantly downregulated under the treatment of tumor necrosis factor-alpha (TNF-α). Furthermore, overexpression of CAR markedly prevented TNF-α induced inflammatory response, TJs dysregulation, and permeability disruption (FITC-Dextran permeability assay) in cells. Consistent with these findings in vitro, we found that CAR overexpression could suppress gut inflammation, attenuate the downregulation of TJ protein ZO-1 and Occludin, and limit the induction of barrier permeability in a DSS induced ulcerative colitis rat model in vivo. Together, our findings strongly suggest that CAR could protect tight junctions and has an anti-inflammatory effect during the pathogenesis of IBD. Thus CAR may serve as a therapeutic target for the diagnosis and treatment of IBD.

SDHB downregulation facilitates the proliferation and invasion of colorectal cancer through AMPK functions excluding those involved in the modulation of aerobic glycolysis

Loss-of-function of succinate dehydrogenase-B (SDHB) is a predisposing factor of aerobic glycolysis and cancer progression. Adenosine monophosphate activated protein kinase (AMPK) is involved in the regulation of aerobic glycolysis and the diverse hallmarks of cancer. The present study investigated whether AMPK mediated the regulatory effects of SDHB in aerobic glycolysis and cancer growth. The expression of SDHB and AMPK in colorectal cancer (CRC) and normal tissues was assessed by western blotting. HT-29 CRC cells were used to establish in vitro models of ectopic overexpression and knockdown of SDHB. SDHB was downregulated, while AMPK and phosphorylated-AMPK (Thr172) were upregulated in CRC tissues. Experiments involving the loss- or gain-of-function of SDHB, revealed that this protein negatively regulated AMPK by influencing its expression and activity. However, SDHB and AMPK were identified to suppress lactic acid production in CRC cells, indicating that each had an inhibitory effect on aerobic glycolysis. Therefore, the regulation of aerobic glycolysis by SDHB is unlikely to be mediated via AMPK. SDHB knockdown promoted the viability, migration and invasion of HT-29 cells, whereas inhibition of AMPK demonstrated the opposite effect. SDHB overexpression impaired cell migration and invasion, and this effect was reversed following AMPK activation. These results indicate that AMPK may mediate the effects of SDHB in CRC cell proliferation and migration. In conclusion, SDHB downregulation in CRC cells may increase AMPK activity, which may subsequently facilitate the proliferation and invasion of these cancer cells. However, the regulation of aerobic glycolysis by SDHB may be independent of AMPK. Further studies are warranted to elucidate the mechanism by which SDHB regulates aerobic glycolysis.

[Effect of hydrogen sulfide on the expression of CSE, NF-κB, and IL-8 mRNA in GES-1 cells with Helicobacter pylori infection].

OBJECTIVEnTo investigate the effect of hydrogen sulfide (H2S) on the expression of CSE, NF-κB, and IL-8 mRNA in GES-1 cells with Helicobacter pylori (H. pylori) infection and to explore its mechanism on gastric mucosa inflammation caused by H. pylori.nnnMETHODSnGES-1 cells were cultured for 24 h and divided into a control group (neither H. pylori nor NaHS), an H. pylori group, a NaHS group (which was further divided into 4 groups at 50, 100, 200, or 400 μmol/L NaHS), and H. pylori + NaHS group (which was further divided into 4 groups at 50, 100, 200, or 400 μmol/L NaHS). Each group was then cultured for 3, 6, or 12 h. The expression of CSE, NF-κB, and IL-8 mRNA was measured by RT-PCR, and their correlation was analyzed.nnnRESULTSnThe expression of CSE, NF-κB, and IL-8 mRNA in GES-1 cells in the H. pylori group was higher than that in the control group. The expression of CSE in the 200 μmol/L NaHS group and 400 μmol/L NaHS group was lower than that of the control group (P<0.05), whereas the expression of NF-κB and IL-8 in all NaHS groups had no statistical differences compared with the control group (P>0.05). The expression of CSE, NF-κB, and IL-8 mRNA in all groups of NaHS, H. pylori + 200 μmol/L NaHS group, and H. pylori + 400 μmol/L NaHS group was lower than that in the H. pylori group (P<0.05). There was positive correlation among the expressions of CSE, NF-κB, and IL-8 mRNA in the H. pylori group, the H. pylori + 200 μmol/L NaHS group, and the H. pylori + 400 μmol/L NaHS group (P<0.05).nnnCONCLUSIONnH. pylori can induce NF-κB and IL-8 mRNA expression and upregulate CSE mRNA expression. At 200 and 400 μmol/L, NaHS can suppress H. pylori-induced NF-κB and IL-8 mRNA expression and ameliorate the morphology of H. pylori-induced GES-1 injury, which may protect gastric epithelial cells by H. pylori infection.

Prokaryotic expression and antigenicity of the CagA gene in Helicobacter pylori

OBJECTIVEnTo synthesize the specific CagA gene segment of the gastric cancer idiotype Helicobacter pylori (H. pylori), establish the prokaryotic expression system and identify the antigenicity sequence of recombination signals.nnnMETHODSnWe selected the CagA fragment which was related to gastric cancer in our earlier research. The CagA gene segment was optimized and synthesized. The synthesized CagA gene was cut from the pUC57-CagA plasmid and then was carried by expression vector pET32a to be transformed into the host bacterium BL21 (DE3). The positively cloned pET32a-CagA was selected by receptivity of aniline and colony PCR. The host bacterium with pET32a-CagA was induced by IPTG to express fusion protein. The expression of CagA protein was analyzed by SDS-PAGE gel electrophoresis and the antigenicity of fusion protein was examined by Western blot.nnnRESULTSnCagA gene segment was designed and synthesized. The sequence of synthesis CagA gene segment was the same as the one designed before (AF289435). We successfully constructed the plasmid of prokaryotic expression of the pET32a-CagA. Homology of the target CagA proteinum was 100%, the same as AAG09884. The host bacterium BL21 (DE3) containing pET32a-CagA could express CagA fusion protein after the IPTG induction. SDS-PAGE gel electrophoresis showed that the molecular weight of fusion protein was the same as expected (45 kD). Western blot showed that the fusion protein could be combined with the antibody of the whole bacterium of anti-H. pylori.nnnCONCLUSIONnThe synthesized CagA fusion protein from the prokaryotic expression system has antigenicity. We hope to set the foundation for selecting the strain in H. pylori correlated to gastric cancer and corresponding therapy in clinical practice.