Kenro Hirata

Overexpression of miR-142-5p and miR-155 in Gastric Mucosa-Associated Lymphoid Tissue (MALT) Lymphoma Resistant to Helicobacter pylori Eradication

microRNAs (miRNAs) are small non-coding RNAs that can function as endogenous silencers of target genes and play critical roles in human malignancies. To investigate the molecular pathogenesis of gastric mucosa-associated lymphoid tissue (MALT) lymphoma, the miRNA expression profile was analyzed. miRNA microarray analysis with tissue specimens from gastric MALT lymphomas and surrounding non-tumor mucosae revealed that a hematopoietic-specific miRNA miR-142 and an oncogenic miRNA miR-155 were overexpressed in MALT lymphoma lesions. The expression levels of miR-142-5p and miR-155 were significantly increased in MALT lymphomas which do not respond to Helicobacter pylori (H. pylori) eradication. The expression levels of miR-142-5p and miR-155 were associated with the clinical courses of gastric MALT lymphoma cases. Overexpression of miR-142-5p and miR-155 was also observed in Helicobacter heilmannii-infected C57BL/6 mice, an animal model of gastric MALT lymphoma. In addition, miR-142-5p and miR-155 suppress the proapoptotic gene TP53INP1 as their target. The results of this study indicate that overexpression of miR-142-5p and miR-155 plays a critical role in the pathogenesis of gastric MALT lymphoma. These miRNAs might have potential application as therapeutic targets and novel biomarkers for gastric MALT lymphoma.

Effective inhibition of cell growth and invasion of melanoma by combined suppression of BRAF (V599E) and Skp2 with lentiviral RNAi

p27Kip1 that regulates the G1/S transition of cell cycle and inhibits Rho A signaling is frequently lost in several cancers leading to the deregulation of cell growth and cell motility. Mitogen‐activated protein kinases (MAPK) regulate the export of p27Kip1 from nucleus to cytoplasm, followed by the degradation with proteases. Skp‐2, a subunit of an SCF ubiquitin‐protein ligase complex responsible for the ubiquitination of p27Kip1, is upregulated frequently in several cancers, leading to the decrease of p27Kip1. We applied human immunodeficiency virus (HIV) lentivirus‐mediated RNA interference (RNAi) to melanoma cells with the BRAF mutation (V599E) and overexpressed Skp‐2 and found that the simultaneous suppression of these activated oncogenes resulted in the effective inhibition of in vitro cell growth and invasive ability of melanoma cells accompanied by the additional increase of p27Kip1. Our results suggest that gene therapy against melanoma with the enhanced MAPK and ubiquitin‐proteasomal pathways could be a specific and effective therapeutic strategy for cancers.

Contribution of efflux pumps to clarithromycin resistance in Helicobacter pylori

Background and Aims:  Although clarithromycin (CLR) is one of the most commonly recommended component drugs of Helicobacter pylori eradication regimens, the prevalence of CLR‐resistant H. pylori has been increasing. It is well known that CLR resistance is associated with point mutations in 23S rRNA, but an active multidrug efflux mechanism of H. pylori may also play a role in its drug resistance. At least four gene clusters have been identified as efflux pump systems in H. pylori and the present study was designed to investigate their role in the CLR resistance of clinical isolates of H. pylori.

CD44 variant 9 expression in primary early gastric cancer as a predictive marker for recurrence

Background:Multiple early gastric cancers (EGCs) may develop in 6–14% of patients even after achieving curative endoscopic submucosal dissection (ESD); however, a useful biomarker for predicting recurrence is not available. The present study investigated whether the expression of CD44 variant 9 (CD44v9), a functional cancer stem cell marker, in the primary gastric cancer tissue represents an indicator of recurrence.Methods:Eighty-eight patients who underwent ESD for EGC from 2008 to 2010 were enrolled and monitored for recurrence for 3 years. The expression levels of CD44v9 in the tissue of initial EGCs were evaluated by immunohistochemistry, and the recurrence rate was compared between CD44v9-positive and CD44v9-negative groups. The mucin phenotype and expression of microRNA-21 (miR-21) and programmed cell death protein 4 (PDCD4) were also analysed.Results:The recurrence rate of EGC was significantly higher in the CD44v9-positive group than in the CD44v9-negative group (hazard ratio (HR), 21.8; 95% confidence interval (CI), 5.71–83.1). However, mucin phenotypes and the expression of miR-21 and PDCD4 did not predict recurrence after ESD. Meanwhile, grade of gastric atrophy was also identified as a significant marker of multiple recurrence (HR, 4.95; 95% CI, 1.30–18.8).Conclusion:CD44 variant 9 expression represents a potential predictive marker for recurrence in EGC.

The tumor suppressor microRNA-29c is downregulated and restored by celecoxib in human gastric cancer cells.

MicroRNAs (miRNAs) are small noncoding RNAs that function as endogenous silencers of target genes and play critical roles during carcinogenesis. The selective cyclooxygenase‐2 (COX‐2) inhibitor celecoxib has been highlighted as a potential drug for treatment of gastrointestinal tumors. The aim of this study was to investigate the role of miRNAs in gastric carcinogenesis and the feasibility of a new therapeutic approach for gastric cancer. miRNA expression profiles were examined in 53 gastric tumors including gastric adenomas (atypical epithelia), early gastric cancers and advanced gastric cancers and in gastric cancer cells treated with celecoxib. miRNA microarray analysis revealed that miR‐29c was significantly downregulated in gastric cancer tissues relative to nontumor gastric mucosae. miR‐29c was significantly activated by celecoxib in gastric cancer cells. Downregulation of miR‐29c was associated with progression of gastric cancer and was more prominent in advanced gastric cancers than in gastric adenomas and early gastric cancer. In addition, expression of the oncogene Mcl‐1, a target of miR‐29c, was significantly increased in gastric cancer tissues relative to nontumor gastric mucosae. Activation of miR‐29c by celecoxib induced suppression of Mcl‐1 and apoptosis in gastric cancer cells. These results suggest that downregulation of the tumor suppressor miR‐29c plays critical roles in the progression of gastric cancer. Selective COX‐2 inhibitors may have clinical promise for the treatment of gastric cancer via restoration of miR‐29c.

Dysfunctional Gastric Emptying With Down-regulation of Muscle-Specific MicroRNAs in Helicobacter pylori-Infected Mice

BACKGROUND & AIMS Little is known about the pathogenic mechanisms of functional dyspepsia. We investigated the role of microRNAs (miRNAs) in gastric motility disorders associated with Helicobacter pylori infection. METHODS Male C57BL/6 mice were infected with H pylori. After long-term infection, gastric emptying was examined and compared with that of uninfected mice (controls). The miRNA expression profile was analyzed by miRNA microarray and quantitative reverse-transcriptase polymerase chain reaction. The results obtained from the animal study were confirmed by in vitro experiments. RESULTS Gastric emptying was significantly accelerated in mice after chronic infection with H pylori. Histologic examination showed that the muscular layers of the stomachs of H pylori-infected mice were significantly thickened. The miRNA expression profile revealed that the muscle-specific miRNAs miR-1 and miR-133 were significantly down-regulated in the stomachs after long-term infection with H pylori. However, expression of histone deacetylase 4 and serum response factor, which are reported target genes of miR-1 and miR-133, increased. Down-regulation of miR-1 and miR-133 and increased cell proliferation were observed in C2C12 mouse myoblast cells after coculture with H pylori. CONCLUSIONS Chronic infection with H pylori down-regulates expression of muscle-specific miRNAs and up-regulates expression of histone deacetylase 4 and serum response factor. These might cause hyperplasia in the muscular layer of the stomach and dysfunction in gastric emptying. These findings provide insight into the molecular pathogenesis of gastric motility disorders, including functional dyspepsia.

Two Amino Acids Mutation of Ferric Uptake Regulator Determines Helicobacter pylori Resistance to Metronidazole

Metronidazole (Mtz) is a prodrug that is converted to its active form when its nitro group is reduced and superoxide radicals are generated. The superoxide radicals are directly toxic to the bacterium. On the other hand, the transcriptional regulator, ferric uptake regulator (Fur), of Helicobacter pylori is a direct suppressor of the iron-cofactored superoxide dismutase SodB, which is essential for protection against superoxide attack. Here, we demonstrate that in some Mtz-resistant strains, SodB activity is induced in a dose-dependent manner on exposure to Mtz. Further, under Mtz exposure, the generation of superoxide radicals in Mtz-resistant strains was significantly reduced as compared with that in the Mtz-susceptible strains. These Mtz-resistant strains were found to carry amino acids mutation of Fur (C78Y, P114S; mutant-type Fur). The binding affinity of the mutant-type Fur to an operator sequence on the sodB promoter (Fur-Box) was significantly reduced. Our approach demonstrated that SodB expression is derepressed by mutant-type Fur, which is associated with the development of Mtz resistance.

Efficacy of sitafloxacin-based rescue therapy for Helicobacter pylori after failures of first- and second-line therapies

ABSTRACT Sitafloxacin-based triple therapy achieved 83.6% (per-protocol) and 78.2% (intention-to-treat) success in eradicating Helicobacter pylori among 78 Japanese patients after clarithromycin-based first-line and metronidazole-based second-line triple therapies failed. Eradication succeeded in 32 out of 43 patients, even with gyrA mutation-positive Helicobacter pylori (per protocol). The position of the gyrA mutation (N87 or D91) was determined to be a better marker than MIC levels for predicting outcomes of sitafloxacin-based treatment.

High frequency of overlap between functional dyspepsia and overactive bladder

Background  Overactive bladder syndrome (OAB) is defined as a symptom complex comprising urgency, with or without urge incontinence, and usually frequency and nocturia. The association between irritable bowel syndrome (IBS) and bladder symptoms has been reported. This study is designed to investigate whether functional dyspepsia (FD), like IBS, is associated with OAB.

Enhanced bacterial efflux system is the first step to the development of metronidazole resistance in Helicobacter pylori.

Although metronidazole (Mtz) is an important component of Helicobacter pylori eradication regimens, it has been pointed out that the increasing use of Mtz may result in increase in the incidence of Mtz-resistant strains. The present study was designed to examine the initial mechanism of resistance acquisition of H. pylori to Mtz. After 10 Mtz-susceptible strains were cultured on plates containing sub-inhibitory concentrations of Mtz, the MIC of Mtz for 9 of the 10 strains increased to levels of the Mtz-resistant strains. In the Mtz-resistance-induced strains, the expression of the TolC efflux pump (hefA) was significantly increased under Mtz exposure, without the reduction of the Mtz-reductive activity. Our finding suggests that overexpression of hefA may be the initial step in the acquisition of Mtz resistance in H. pylori.