Kayoko Matsushima

MicroRNA signatures in Helicobacter pylori‐infected gastric mucosa

The study was conducted to determine expression patterns of microRNA (miRNA), a non‐coding RNA that controls gene expression mainly through translational repression, in gastric mucosa infected with Helicobacter pylori. Using endoscopic biopsy specimens, miRNA expression patterns in H. pylori‐infected gastric mucosa were determined by microarray. The differentially expressed miRNAs were quantitated by real‐time reverse‐transcriptase polymerase chain reaction (RT‐PCR). An in vitro infection model was assessed to monitor the regulation of miRNAs in gastric epithelium in response to H. pylori. The comprehensive method unraveled the expression profiles; among 470 human miRNAs loaded, 55 were differentially expressed between H. pylori‐positive and ‐negative subjects. The expression levels were significantly decreased in 30 miRNAs, whereas hsa‐miRNA‐223 was the only miRNA to be overexpressed on quantitative RT‐PCR. Eight miRNAs enabled discrimination of H. pylori status with acceptable accuracy. Gastritis scores of activity and chronic inflammation according to the updated Sydney system correlated significantly with the expression levels of diverse miRNAs. Cure of the infection with an anti‐H. pylori regimen restored decreased expression in 14 of the 30 miRNAs. Expression levels of some miRNAs, including let‐7 family members, were significantly altered following infection with a virulent H. pylori strain carrying intact cag pathogenicity island including cagA but not isogenic mutants. These results provide insights into miRNA involvement in the pathogenesis of H. pylori‐associated gastritis. cagA may be involved in cellular regulation of certain miRNAs in the gastric epithelium.

MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.MethodsTotal RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.ResultsBased on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.ConclusionsThese results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.

Peroral endoscopic myotomy for esophageal achalasia: Clinical impact of 28 cases

The aim of the present study was to clarify the efficacy of peroral endoscopic myotomy (POEM) for esophageal achalasia.

MicroRNAs and Esophageal Squamous Cell Carcinoma

Esophageal cancer is a common cause of cancer death worldwide. Esophageal squamous cell carcinoma (ESCC) is the most predominant type. Certain microRNAs (miRNAs) function in tumorgenesis involved in important biological and pathologic processes. To reveal miRNAs’ signatures of ESCC, we analyzed miRNAs extracted from ESCC cell lines with the microRNA microarray. The significant alterations were confirmed by quantitative real-time PCR using miRNAs extracted from cell lines or patients’ esophageal biopsy tissues. We found that miR-205 and miR-10a were significantly altered in cellular expression, and might be specific for ESCC with potential roles in the pathogenesis. As a result of function studies in miR-205, alterations in miR-205 expression could modulate the phenotype of epithelial cells towards epithelial-mesenchymal transition characterized by reduced abundance of E-cadherin, that is the ESCC-specific miRNA target and inhibit translationally a representative E-cadherin repressor, ZEB1 and ZEB2. Similarly, miR-10a is reported as a tumor suppressor by controlling cell migration/invasion, as it can target homeobox genes. These results provide insight into the potential mechanisms of ESCC in the pathogenesis. This review also includes a comprehensive overview of the relationship between miRNAs and ESCC.

Magnifying Endoscopic Findings Can Predict Clinical Outcome during Long-Term Follow-Up of More Than 12 Months in Patients with Ulcerative Colitis

Background and Aims. To explore the association of magnifying endoscopic (ME) findings with histopathology and relapse in ulcerative colitis (UC). Methods. Forty-six patients with UC underwent ME with narrow band imaging (NBI) and crystal violet staining and were followed for more than 12 months. ME findings with vital staining were classified into ME-A, regular arrangement of round to oval pits; ME-B, irregular arrangement with/without enlarged spaces between even pits; ME-C, irregular pits in size and shape with more irregular arrangement of pits; and ME-D, disrupted or disappeared pits. NBI-guided ME features of microvascular pattern (MVP) were divided into the MVP-regular and MVP-irregular type. Results. There were 5, 24, 10, and 7 cases of ME-A, ME-B, ME-C, and ME-D grade, respectively, while there were 21 and 25 of MVP-regular and MVP-irregular type, respectively. ME classifications were significantly associated with Matts endoscopic grade. ME classifications and MVP types were significantly associated with each pathognomonic microscopic feature of severe mucosal inflammation, crypt abscess, and goblet cell depletion. There were significant differences in the percentages of remission among ME classifications and between MVP types. Conclusion. ME findings can be predictive of relapse in UC and reliable for in vivo histopathological assessment.

Helicobacter pylori VacA Reduces the Cellular Expression of STAT3 and Pro-survival Bcl-2 Family Proteins, Bcl-2 and Bcl-XL, Leading to Apoptosis in Gastric Epithelial Cells

BackgroundHelicobacter pylori vacuolating cytotoxin, VacA, stimulates apoptosis via a mitochondria-dependent pathway. VacA induces apoptosis via activation of the pro-apoptotic B-cell lymphoma (Bcl)-2 family proteins, Bcl-2-associated X protein (Bax) and Bcl-2 homologous antagonist/killer (Bak), while the implication of such pro-survival Bcl-2 family members as Bcl-2 and Bcl-XL in the VacA-induced apoptosis remains unknown. Signal transduction and activator of transcription 3 (STAT3) is a pivotal transcription factor that upregulates Bcl-2 and Bcl-XL.AimsThis study was conducted to elicit the implication of STAT3 and pro-survival Bcl-2 and Bcl-XL in the intrinsic apoptosis.MethodsImmunoblot and reverse transcriptase real-time polymerase chain reaction (RT-PCR) were employed to assess the cellular expression of STAT3, Bcl-2, and Bcl-XL in response to purified VacA in gastric adenocarcinoma cell lines. VacA-induced apoptosis was quantitated morphologically following knockdown by each specific small interfering RNA (siRNA) or in the presence of pharmacological inhibitors.ResultsVacA reduced STAT3, Bcl-2, and Bcl-XL expression in a dose-dependent manner. Knockdown of STAT3, Bcl-2, and Bcl-XL by siRNA induced apoptosis to a similar extent in the case of sufficient VacA inoculation. The VacA-mediated reduction of STAT3 expression was independent of cellular vacuolization, since a vacuolar-type ATPase inhibitor, bafilomycin A1, did not inhibit VacA-induced reduction of STAT3, Bcl-2, and Bcl-XL expression. Instead, a c-JUN NH2-terminal kinase (JNK) inhibitor, SP600125, restored the VacA-induced reduction of STAT3 expression to the basal level.ConclusionsVacA-induced apoptosis may be, in part, implicated in the reduction of STAT3 linking to the downregulation of Bcl-2 and Bcl-XL, in association with JNK activity.

Per-oral endoscopic myotomy: Emerging indications and evolving techniques

Esophageal achalasia is a benign esophageal motility disorder resulting from an impaired relaxation of the lower esophageal sphincter. The principles of treatment involve disruption of the sphincter at the esophagogastric junction. Treatment techniques include balloon dilatation, botulinum toxin injection, and surgical myotomy. In 2008, per‐oral endoscopic myotomy (POEM) was introduced by Inoue et al. as an endoscopic myotomy with no skin incision. The procedure has been well accepted and widely applied owing to its minimal invasiveness and high cure rates. Moreover, there have been discussions on wider indications for POEM and new technical developments have been reported. The present article reviews the historical background and present status of POEM, as well as future prospects for its application in the treatment of esophageal achalasia.

Endoplasmic Reticulum Stress Contributes to Helicobacter Pylori VacA-Induced Apoptosis

Vacuolating cytotoxin A (VacA) is one of the important virulence factors produced by H. pylori. VacA induces apoptotic cell death, which is potentiated by ammonia. VacA also causes cell death by mitochondrial damage, via signaling pathways that are not fully defined. Our aim was to determine whether endoplasmic reticulum (ER) stress is associated with VacA-induced mitochondrial dysfunction and apoptosis. We found that C/EBP homologous protein (CHOP), a key signaling protein of ER stress-induced apoptosis, was transcriptionally up-regulated following incubation of gastric epithelial cells with VacA. The effect of VacA on CHOP induction was significantly enhanced by co-incubation with ammonium chloride. Phosphorylation of eukaryotic initiation factor 2 (eIF2)-alpha, which is known to occur downstream of the ER stress sensor PKR-like ER-localized eIF2-alpha kinase (PERK) and to regulate CHOP expression, was also observed following incubation with VacA in the presence of ammonium chloride. Knockdown of CHOP by siRNA resulted in inhibition of VacA-induced apoptosis. Further studies showed that silencing of the PERK gene with siRNA attenuated VacA-mediated phosphorylation of eIF2-alpha, CHOP induction, expression of BH3-only protein Bim and Bax activation, and cell death induced by VacA with ammonium chloride, indicating that ER stress may lead to mitochondrial dysfunction during VacA-induced toxicity. Activation of ER stress and up-regulation of BH3-only proteins were also observed in human H. pylori-infected gastric mucosa. Collectively, this study reveals a possible association between VacA-induced apoptosis in gastric epithelial cells, and activation of ER stress in H. pylori-positive gastric mucosa.

Early and long-term outcomes of endoscopic submucosal dissection for early gastric cancer in a large patient series.

Endoscopic submucosal dissection (ESD) enables the curative resection of early gastric cancer (EGC); however, little information is available on the long-term outcomes of ESD. This study was conducted to clarify the clinical outcomes of a large number of patients with EGC who underwent ESD. The early outcomes were assessed in 1,209 patients and the long-term outcomes were assessed in 300 patients at a follow-up >5 years after the ESD procedure. The overall survival rates were compared between indication and expanded-indication groups, and between the patients who did or did not undergo additional surgery in an out-of-indication group. Overall survival rates were also compared among different age groups. In total, 617 lesions were classed as the indication group, 507 as the expanded-indication group and 208 as the out-of-indication group. Curative resection rates were 96.6% and 91.5% in the indication and expanded-indication groups, respectively. In terms of the long-term outcomes, 20 of the 146 patients in the indication group, 15 of the 105 patients in the expanded-indication group and one of the 23 patients who underwent additional surgery in the out-of-indication group succumbed due to causes other than gastric cancer. Among the 26 patients who did not undergo additional surgery in the out-of-indication group, 10 mortalities occurred, including one due to gastric cancer. The five-year survival rates were not significantly different between the indication and expanded-indication groups. In the out-of-indication group, the five-year survival rate for the patients who did not undergo additional surgery (65.0%) was significantly lower than that for those who did undergo additional surgery (100%) (P<0.01). The five-year survival rate of patients aged >80 years (67.1%) was significantly lower than that of the younger patients (<60 years, 91.6%; sixties, 93.0%; seventies, 84.5%) (P<0.0001). In conclusion, although expanded-indication of ESD for EGC is appropriate, comorbidities require consideration in elderly patients.

Enhanced expression of CCL20 in human Helicobacter pylori-associated gastritis

CC chemokine ligand 20 (CCL20) attracts CC chemokine receptor 6 (CCR6)-expressing cells. Using endoscopic biopsies taken from the gastric antrum of 42 subjects infected with H. pylori and 42 uninfected subjects, mucosal CCL20 mRNA and protein levels were measured by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. CCL19 mRNA and protein levels, as well as CCL21 mRNA levels, were also measured. The CCL20 mRNA and protein levels were significantly elevated in H. pylori-positive patients and substantially decreased after successful eradication. CCL19 and CCL21 expression levels were comparable in the H. pylori-infected and the uninfected groups. The CCL20 concentrations correlated with the degree of chronic gastritis. Immunohistochemistry and the in vitro infection assay showed that CCL20 was principally produced by the gastric epithelium. CCR6-expressing cells, including CD45RO(+) memory T lymphocytes and fascin(+)-CD1a(+) immature dendritic cells, infiltrated close to the CCL20-expressing epithelial cells. The CCL20/CCR6 interaction may be involved in the development of H. pylori-associated gastritis.