Role of microRNAs in gastric cancer

Although gastric cancer(GC)is one of the leading causes of cancer-related death,major therapeutic advances have not been made,and patients with GC still face poor outcomes.The prognosis of GC also remains poor because the molecular mechanisms of GC progression are incompletely understood.MicroRNAs(miRNAs)are noncoding RNAs that are associated with gastric carcinogenesis.Studies investigating the regulation of gene expression by miRNAs have made considerable progress in recent years,and abnormalities in miRNA expression have been shown to be associated with the occurrence and progression of GC.miRNAs contribute to gastric carcinogenesis by altering the expression of oncogenes and tumor suppressors,affecting cell proliferation,apoptosis,motility,and invasion.Moreover,a number of miRNAs have been shown to be associated with tumor type,tumor stage,and patient survival and therefore may be developed as novel diagnostic or prognostic markers.In this review,we discuss the involvement of miRNAs in GC and the mechanisms through which they regulate gene expression and biological functions.Then,we review recent research on the involvement of miRNAs in GC prognosis,their potential use in chemotherapy,and their effects on Helicobacter pylori infections in GC.A greater understanding of the roles of miRNAs in gastric carcinogenesis could provide insights into the mechanisms of tumor development and could help to identify novel therapeutic targets.     

Role of protease-activated receptors in the vascular system

Thrombin is one of the key molecules involved in the development of vascular diseases. Thrombin does not only serve as a coagulation factor, but it also exerts cellular effects by activating protease (proteinase)-activated receptors (PARs), a family of seven-transmembrane G protein-coupled receptors. This study focused on the role of PARs in the vascular system. Among the four members so far identified, PAR-1 and PAR-2 were found to play an important role in the vascular system, while the functional roles of PAR-3 and PAR-4 appear to be mostly limited to platelets. The endothelial cells play a primary role in mediating the vascular effects of PARs under physiological conditions, while PARs of the smooth muscle cells can be induced under pathological conditions, and therefore play a more pathophysiological role. PAR-1 and PAR-2 mediate various vascular effects including regulation of vascular tone, proliferation and hypertrophy of smooth muscle and angiogenesis. Since proteases are activated under pathological conditions such as hemorrhage, tissue damage, and inflammation, PARs are suggested to play a critical role in the development of functional and structural abnormality in the vascular lesion. Understanding the functional role of PARs in the vascular system can thus help in the development of new strategies for the prevention and therapy of vascular diseases.     

Helicobacter pylori infection,gastrin and cyclooxygenase-2 in gastric carcinogenesis

Gastric cancer is one of the most frequent neoplasms and a main cause of death worldwide, especially in China and Japan. Numerous epidemiological, animal and experimental studies support a positive association between chronic Helicobacter pylori(H. pylori) infec-tion and the development of gastric cancer. However, the exact mechanism whereby H. pylori causes gastric carcinogenesis remains unclear. It has been demon-strated that expression of cyclooxygenase-2(COX-2) is elevated in gastric carcinomas and in their precursor le-sions. In this review, we present the latest clinical and experimental evidence showing the role of gastrin and COX-2 in H. pylori-infected patients and their possible association with gastric cancer risk.     

Emerging roles of protease-activated receptors in cardiometabolic disorders

Cardiometabolic disorders, including obesity-related insulin resistance and atherosclerosis, share sterile chronic inflammation as a major cause; however, the precise underlying mechanisms of chronic inflammation in cardiometabolic disorders are not fully understood. Accumulating evidence suggests that several coagulation proteases, including thrombin and activated factor X (FXa), play an important role not only in the coagulation cascade but also in the proinflammatory responses through protease-activated receptors (PARs) in many cell types. Four members of the PAR family have been cloned (PAR 1–4). For instance, thrombin activates PAR-1, PAR-3, and PAR-4. FXa activates both PAR-1 and PAR-2, while it has no effect on PAR-3 or PAR-4. Previous studies demonstrated that PAR-1 and PAR-2 activated by thrombin or FXa promote gene expression of inflammatory molecules mainly via the NF-κB and ERK1/2 pathways. In obese adipose tissue and atherosclerotic vascular tissue, various stresses increase the expression of tissue factor and procoagulant activity. Recent studies indicated that the activation of PARs in adipocytes and vascular cells by coagulation proteases promotes inflammation in these tissues, which leads to the development of cardiometabolic diseases. This review briefly summarizes the role of PARs and coagulation proteases in the pathogenesis of inflammatory diseases and describes recent findings (including ours) on the potential participation of this system in the development of cardiometabolic disorders. New insights into PARs may ensure a better understanding of cardiometabolic disorders and suggest new therapeutic options for these major health threats.     

Coagulation,Protease-Activated Receptors,and Viral Myocarditis

The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling, and heart failure. A recent study using a mouse model have shown that tissue factor, thrombin, and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new strategy to reduce viral myocarditis.     

Regulation of protease-activated receptor-1 in mononuclear cells by neutrophil proteases

Neutrophils and mononuclear cells are implicated in the pathogenesis of several inflammatory conditions including chronic obstructive pulmonary disease (COPD). Neutrophil-derived serine proteases, such as cathepsin G (CG) and neutrophil elastase (NE), may interact with mononuclear cells via protease-activated receptors (PARs) which are seven-transmembrane G protein-coupled receptors activated by proteolytic cleavage of the extracellular N-terminus, and which, on activation, induce the release of several mediators and cytokines. We determined whether CG and NE could affect PAR-1 expression and function in mononuclear cells. Human blood mononuclear cells were isolated from 20 healthy donors. Surface and intracellular receptor expression and calcium mobilisation (using the calcium chelator, FLUO3-AM) were studied by fluorescence-assisted cell sorting (FACS analysis). Positive controls, i.e. thrombin (0.1-100 mU/ml) and the PAR-1-activating peptide SFLLRN (100 microM) induced a rapid and transient intemalisation of PAR-1 in monocytes and lymphocytes. CG but not NE had a similar effect. By contrast, in monocytes intracellular calcium mobilisation was induced by thrombin and SFLLRN but not by CG and NE. Thus, CG can induce intracellular PAR-1 sequestration without activation of the receptor, and may act as an antagonist and prevent subsequent activation of PAR-1 in mononuclear cells. These findings may be of relevance to the pathogenesis of COPD.     

Epigenetic regulation of DNA repair machinery in Helicobacter pylori-induced gastric carcinogenesis

Although thousands of DNA damaging events occur in each cell every day, efficient DNA repair pathways have evolved to counteract them. The DNA repair machinery plays a key role in maintaining genomic stability by avoiding the maintenance of mutations. The DNA repair enzymes continuously monitor the chromosomes to correct any damage that is caused by exogenous and endogenous mutagens. If DNA damage in proliferating cells is not repaired because of an inadequate expression of DNA repair genes, it might increase the risk of cancer. In addition to mutations, which can be either inherited or somatically acquired, epigenetic silencing of DNA repair genes has been associated with carcinogenesis. Gastric cancer represents the second highest cause of cancer mortality worldwide. The disease develops from the accumulation of several genetic and epigenetic changes during the lifetime. Among the risk factors, Helicobacter pylori (H. pylori) infection is considered the main driving factor to gastric cancer development. Thus, in this review, we summarize the current knowledge of the role of H. pylori infection on the epigenetic regulation of DNA repair machinery in gastric carcinogenesis.     

A protective role of protease-activated receptor 1 in rat gastric mucosa

BACKGROUND AND AIMS: On activation, protease-activated receptor (PAR)-2 modulates multiple gastric functions and exerts mucosal protection via activation of sensory neurons. The role of PAR-1, a thrombin receptor, in the stomach remains unknown. We thus examined if the PAR-1 agonist could protect against gastric mucosal injury in rats. METHODS: Gastric mucosal injury was created by oral administration of ethanol/HCl or absolute ethanol in conscious rats. Gastric mucosal blood flow and acid secretion were determined in anesthetized rats. Immunohistochemical analyses of PAR-1 and cyclooxygenase (COX)-1 were also performed in rat and human stomach. RESULTS: The PAR-1 agonist TFLLR-NH(2), administered intravenously in combination with amastatin, protected against the gastric mucosal injury induced by ethanol/HCl or absolute ethanol. The protective effect of TFLLR-NH(2) was abolished by indomethacin or a COX-1 inhibitor but not by ablation of sensory neurons with capsaicin. TFLLR-NH(2) produced an NO-independent increase in gastric mucosal blood flow that was partially inhibited by blockade of the endothelium-derived hyperpolarizing factor pathway. This inhibitory effect was promoted by indomethacin. TFLLR-NH(2) suppressed carbachol-evoked acid secretion in an indomethacin-reversible manner. Immunoreactive PAR-1 and COX-1 were expressed abundantly in rat gastric muscularis mucosae and smooth muscle, and the former protein was also detectable in blood vessels. Similar staining was observed in human gastric muscularis mucosae. CONCLUSIONS: The PAR-1 agonist, given systemically, protects against gastric mucosal injury via COX-1-dependent formation of prostanoids, modulating multiple gastric functions. Our data identify a novel protective role for PAR-1 in gastric mucosa, and the underlying mechanism is entirely different from that for PAR-2.     

Role of the tumor microenvironment in the pathogenesis of gastric carcinoma

Gastric carcinoma(GC)is the 4thmost prevalent cancer and has the 2ndhighest cancer-related mortality rate worldwide.Despite the incidence of GC has decreased over the past few decades,it is still a serious health problem.Chronic inflammatory status of the stomach,caused by the infection of Helicobacter pylori(H.pylori)and through the production of inflammatory mediators within the parenchyma is suspected to play an important role in the initiation and progression of GC.In this review,the correlation between chronic inflammation and H.pylori infection as an important factor for the development of GC will be discussed.Major components,including tumor-associated macrophages,lymphocytes,cancer-associated fibroblasts,angiogenic factors,cytokines,and chemokines of GC microenvironment and their mechanism of action on signaling pathways will also be discussed.Increasing our understanding of how the components of the tumor microenviroment interact with GC cells and the signaling pathways involved could help identify new therapeutic and chemopreventive targets.     

Proteinase-activated receptor 2 is an anti-inflammatory signal for colonic lamina propria lymphocytes in a mouse model of colitis

The proteinase-activated receptor 2 (PAR-2) is a member of a family of G protein-coupled receptors for proteases. Proteases cleave PARs within the extracellular N-terminal domains to expose tethered ligands that bind to and activate the cleaved receptors. PAR-2 is highly expressed in colon in epithelial and neuronal elements. In this study we show that PAR-2 activation prevents the development and induces healing of T helper cell type 1-mediated experimental colitis induced by intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. A role for PAR-2 in the protection against colon inflammation was explored by the use of SLIGRL-NH(2), a synthetic peptide that corresponds to the mouse tethered ligand exposed after PAR-2 cleavage. TNBS-induced colitis was dose-dependently reduced by the administration of SLIGRL-NH(2), whereas the scramble control peptide, LSIGRL-NH(2), was uneffective. This beneficial effect was reflected by increased survival rates, improvement of macroscopic and histologic scores, decrease in mucosal content of T helper cell type 1 cytokines, protein, and mRNA, and a diminished myeloperoxidase activity. SLIGRL-NH(2), but not the scramble peptide, directly inhibited IFN-gamma secretion and CD44 expression on lamina propria T lymphocytes. Protection exerted by PAR-2 in TNBS-treated mice was reverted by injecting mice with a truncated form of calcitonin gene-related peptide and by sensory neurons ablation with the neurotoxin capsaicin. Collectively, these studies show that PAR-2 is an anti-inflammatory receptor in the colon and suggest that PAR-2 ligands might be effective in the treatment of inflammatory bowel diseases.     

Helicobacter pylori associated gastric intestinal metaplasia: Treatment and surveillance

Gastric cancer(GC) is one of the leading causes of cancer related death in the world, particularly in East Asia. According to the Correa's cancer cascade, noncardia GC is usually developed through a series of mucosal changes from non-atrophic gastritis to atrophic gastritis(AG), intestinal metaplasia(IM), dysplasia and adenocarcinoma. Atrophic gastritis and IM are therefore generally considered to be pre-neoplastic gastric lesions. Helicobacter pylori(H. pylori) infection is an important initiating and promoting step of this gastric carcinogenesis cascade. Emerging long-term data showed that eradication of H. pylori reduced the risk of subsequent cancer development. It however remains confusing whether eradication of the bacterium in individuals with pre-neoplastic gastric lesions could regress these changes as well as in preventing cancer. Whilst H. pylori eradication could likely regress AG, the presence of IM may be a point of no return in this cascade. Hence, surveillance by endoscopy may be indicated in those with extensive IM or those with incomplete IM, particularly in populations with high GC risk. The optimal interval and the best tool of surveillance endoscopy remains to be determined in future studies.     

A CD4+ proliferation of large granular lymphocytes expresses the protease activated receptor-1

The platelet-type thrombin receptor was the first member to be identified in a family of protease activated receptors (PARs) and has been designated PAR-1. We recently reported that the large granular lymphocytes (LGLs) in patients with proliferations of CD8⁺ cells co-expressed PAR-1 and the expression of PAR-1 correlated with the expression of CD57. Here we show, by three-colour immunofluorescence, that the LGLs from a patient with a rare CD4⁺ CD57⁺ monoclonal expansion also expressed PAR-1. Northern blot analysis confirmed the presence of high levels of mRNA for PAR-1 in these LGLs.     

Design, synthesis, and biological characterization of a peptide-mimetic antagonist for a tethered-ligand receptor

Protease-activated receptors (PARs) represent a unique family of seven-transmembrane G protein-coupled receptors, which are enzymatically cleaved to expose a truncated extracellular N terminus that acts as a tethered activating ligand. PAR-1 is cleaved and activated by the serine protease alpha-thrombin, is expressed in various tissues (e.g., platelets and vascular cells), and is involved in cellular responses associated with hemostasis, proliferation, and tissue injury. We have discovered a series of potent peptide-mimetic antagonists of PAR-1, exemplified by RWJ-56110. Spatial relationships between important functional groups of the PAR-1 agonist peptide epitope SFLLRN were employed to design and synthesize candidate ligands with appropriate groups attached to a rigid molecular scaffold. Prototype RWJ-53052 was identified and optimized via solid-phase parallel synthesis of chemical libraries. RWJ-56110 emerged as a potent, selective PAR-1 antagonist, devoid of PAR-1 agonist and thrombin inhibitory activity. It binds to PAR-1, interferes with PAR-1 calcium mobilization and cellular function (platelet aggregation; cell proliferation), and has no effect on PAR-2, PAR-3, or PAR-4. By flow cytometry, RWJ-56110 was confirmed as a direct inhibitor of PAR-1 activation and internalization, without affecting N-terminal cleavage. At high concentrations of alpha-thrombin, RWJ-56110 fully blocked activation responses in human vascular cells, albeit not in human platelets; whereas, at high concentrations of SFLLRN-NH(2), RWJ-56110 blocked activation responses in both cell types. Thus, thrombin activates human platelets independently of PAR-1, i.e., through PAR-4, which we confirmed by PCR analysis. Selective PAR-1 antagonists, such as RWJ-56110, should serve as useful tools to study PARs and may have therapeutic potential for treating thrombosis and restenosis.     

Progress in the understanding of protease-activated receptors

Thrombin results from the activation of the blood coagulation system. It is a multifunctional protein that has, besides its function in hemostasis and thrombosis, several cellular effects that link the coagulation system with the inflammatory response. Many years of investigations were necessary for the discovery of the first functional thrombin receptor, which was found to have a unique mechanism of activation. The receptor was named protease-activated receptor 1 (PAR-1) because proteolysis is necessary for its activation. Subsequent studies led to the identification of the other PARs, PAR-2, PAR-3, and PAR-4. PAR-2 is activated by trypsin, tryptase, factor Xa, or factor VIIa, but it cannot be activated by thrombin, PAR-3 and PAR-4 can also be activated by thrombin. Activation of PARs by protease involves proteolytic cleavage and unmasking of an amino-terminal receptor sequence, which acts as a tethered ligand by binding to the second extracellular loop of the receptor to initiate transmembrane signaling. Sequence analysis has shown that all PARs are members of the 7-transmembrane domain receptor superfamily. Expression of PARs has been detected in most tissues and in numerous cells, and thus these molecules have been implicated in several physiological processes and in the pathogenesis of several diseases.     

IL－6对肥大细胞蛋白酶激活受体表达的影响

目的探讨IL-6对P815肥大细胞蛋白酶激活受体（PAR）表达的影响及其在过敏与炎症中的作用机制。方法P815肥大细胞激发后,收集各时间点的细胞和上清。采用实时定量PCR和流式细胞术的方法在mRNA和蛋白水平检测PAR－1、PAR-2、PAR．3和PAR-4表达情况。结果IL-6上调PAR－1、PAR-2、PAR－3、PAR-4mRNA的表达;下调PAR－1、PAR－3、PAR-4蛋白的表达;IL-6抗体的应用可阻断IL-6对PARs表达的上调作用。结论IL-6能够调节肥大细胞表面PAR的表达,并可能通过这一途径参与过敏性炎症反应。     

Helicobacter pylori associated chronic gastritis,clinical syndromes,precancerous lesions,and pathogenesis of gastric cancer development

Helicobacter pylori(H.pylori)infection is well known to be associated with the development of precancerous lesions such as chronic atrophic gastritis(AG),or gastric intestinal metaplasia(GIM),and cancer.Various molecular alterations are identified not only in gastric cancer(GC)but also in precancerous lesions.H.pylori treatment seems to improve AG and GIM,but still remains controversial.In contrast,many studies,including meta-analysis,show that H.pylori eradication reduces GC.Molecular markers detected by genetic and epigenetic alterations related to carcinogenesis reverse following H.pylori eradication.This indicates that these changes may be an important factor in the identification of high risk patients for cancer development.Patients who underwent endoscopic treatment of GC are at high risk for development of metachronous GC.A randomized controlled trial from Japan concluded that prophylactic eradication of H.pylori after endoscopic resection should be used to prevent the development of metachronous GC,but recent retrospective studies did not show the tendency.Patients with precancerous lesions(molecular alterations)that do not reverse after H.pylori treatment,represent the"point of no return"and may be at high risk for the development of GC.Therefore,earlier H.pylori eradication should be considered for preventing GC development prior to the appearance of precancerous lesions.     

CD24 genetic variants contribute to overall survival in patients with gastric cancer

AIM: To investigate the role of single nucleotide polymorphisms(SNPs) in CD24 gene in susceptibility and overall survival of gastric cancer(GC).METHODS: We genotyped 3 tagging SNPs of CD24-P-534 in the promoter region, P170 in the coding region of exon 2 and P1527 in the 3′ untranslated region- using polymerase chain reaction-restriction fragment length polymorphism in specimens from 679 histologically-confirmed GC cases, 111 gastric atrophy(GA) cases and 976 tumor-free controls. Serumimmunoglobulin G antibodies to Helicobacter pylori(H. pylori) of all subjects were detected by enzyme-linked immunosorbent assay. CD24 expression was evaluated by immunohistochemistry in 131 GC specimens. Correlations between SNPs and risk of GC or GA were shown by P values and odd ratios(ORs) with 95% confidence intervals(95%CI) compared with the most common genotype of each SNP using the unconditional logistic regression model after adjusting for age, sex and H. pylori infection. Survival within each SNP group was plotted by Kaplan-Meier method and compared by log-rank test(recessive model). Hazard ratios with 95%CIs were computed by Cox regression model after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy.RESULTS: All of the three loci were in Hardy-Weinberg equilibrium in the control group. Median followup time for the 600 GC patients included in the survival analysis was 36.2 mo(range, 2.1-66.7 mo; 95%CI: 34.3-36.5 mo). Patients with the P-534 A/A genotype had significantly shorter survival(HR = 1.38, 95%CI: 1.01-1.88, P = 0.042) than did the C/C or C/A genotype carriers after adjusting for age, sex, histological type, tumor differentiation, clinical stage and post-operational chemotherapy. This trend was more evident in patients who lived longer than 2.5 years(HR = 7.55, 95%CI: 2.16-26.32, P = 0.001). The P170 T/T genotype was associated with a shorter lifespan than the non-T/T genotypes, but not significantly so. None of the three genetic variants was found to be associated with risk of GC(including tumor stage, grade and distant metastasis) or with risk of gastric atrophy. Furthermore, no difference of CD24 expression was found among the genotypes.CONCLUSION: The P-534 site in CD24 gene affects the overall survival of gastric cancer and may serve as a prognostic marker for gastric cancer.     

Serine proteases excite myenteric neurons through protease-activated receptors in guinea pig small intestine

BACKGROUND & AIMS: Serine proteases are postulated to influence gastrointestinal function by stimulating protease-activated receptors (PARs). This study identified the effects on myenteric neurons of activating PARs and investigated underlying mechanisms of action. METHODS: Intracellular electrophysiologic methods were used to study the effects of proteases on electrical and synaptic behavior of morphologically identified neurons in the guinea pig enteric nervous system. Fluorescent immunohistochemistry was used to study the chemical coding of neurons that responded to PARs stimulation. RESULTS: Application of thrombin, trypsin, or mast cell tryptase evoked slowly activating excitatory responses reminiscent of slow synaptic excitation in enteric neurons. Synthetic activating peptides for PAR-1, -2, and -4 receptors mimicked the actions of the proteases. The depolarizing responses evoked by PARs were insensitive to cyclooxygenase inhibitors and were suppressed by agents that inhibit phospholipase C (PLC) or block intraneuronal receptors for inositol triphosphate. A majority of PAR-sensitive uniaxonal neurons expressed immunoreactivity for nitric oxide synthase. Most of the PAR-sensitive AH Dogiel morphologic type II neurons were immunoreactive for calbindin. CONCLUSIONS: Excitatory responses to the serine proteases are mediated by PAR-1, -2, and -4 receptors. The mechanism of signal transduction involves stimulation of PLC and intraneuronal calcium mobilization and is independent of prostanoid formation.     

Novel role of toll-like receptors in Helicobacter pylori-induced gastric malignancy

Helicobacter pylori(H.pylori)infects the human stomach during infancy and develops into chronic activeinflammation.The majority of H.pylori tend to colonize within the mucous gel layer of the stomach.Thestomach lacks its own immune function,thus innateimmunity as the first line of defense is vital for specificimmunity against H.pylori.We review recent discoveries in the pathophysiologic roles of toll-like receptors(TLRs),mainly TLR2 and TLR4,in H.pylori-induced inflammation.In addition,the TLR pathways activated byH.pylori-induced inflammation have been shown to beclosely associated not only with gastric carcinogenesis,but also with formation of the tumor microenvironmentthrough the production of pro-inflammatory cytokines,chemokines,and reactive oxygen species.Althoughthe correlation between single nucleotide polymorphisms of TLRs and gastric cancer risk remains unclear,a recent study demonstrated that STAT3-driven upregulation of TLR2 might promote gastric tumorigenesis independent of inflammation.Further research onthe regulation of TLRs in H.pylori-associated gastriccarcinogenesis will uncover diagnostic/predictive biomarkers and therapeutic targets for gastric cancer.