Effect Of FoxO3a Signal Network On Helicobacter Pylori Infection

Research Background:Among all the of cancer-related death around the world, gastric cancer which isregarded as the most common malignant tumor in the Gastroenterology Departmentranks in the second place. A lot of researches revealed that the Helicobacter pyloriinfection is closely associated gastric cancer. Helicobacter pylori has been classifiedby World Health Organization as a type1carcinogen with associations to thedevelopment of gastric cancer。Except it, More than50%of the world’s populationharbor H. pylori in their upper gastrointestinal tract. At present, we know there isinseparable relationship between helicobacter pylori and variety of the stomach andduodenum enteric diseases, but which is the exact pathogenic mechanism is not yetclear.Forkhead (Fox) protein family is officially launched and definitely named inyear2000. Now they have been grouped in17subfamilies in which the FoxO is themost deep-studied.FoxO plays a paramount role in the cell apoptosis, the cell cycleblock, DNA repair and oxidative reaction by the transcriptional regulation of thedownstream target genes such as Bim, p27, CyclinD1transcription, etc. Thepreliminary research shows that Hp can activate the PI3K-Akt signaling pathways inthe GES-1cells of normal gastric mucosa epithelial cell line and then cause thedisorders of cell proliferation and apoptosis. FoxO3a as a downsteam target of AKT,it have not been elucidated how Hp impact FoxO3a。So, this study was undertaken toinvestigate the alteration of FoxO3a transcription factors in Helicobacter pylori-related gastric epithelium disorders and the impacts how Helicobacter pylori work onFoxO3a and its downstream targets, such as Bim, p27, CycinD.Research Objectives:1.To study the change rules of FoxO3a transcription factors in the gastricmucous membrane cancerous process of atrophic gastritis-intestinal metaplasia-atypical hyperplasia-gastric cancer by means of the immunohistochemistry methods, and then discuss the clinical significance.2.Respectively, put HP which are of diffferent concentrations in the GES-1cellsfor different timing. Obeserve what is the influence on the survival rate of growth ofcells by Hp.And from the translation of protein expression level and mRNA leveldetection FoxO3a、pFoxO3a、p27、 CyclinD1and Bim expression.Discuss theinfluence on FoxO3a signal network by the Helicobacter pylori infection.Research Methods:1. The part of in-vivo studySelect149pieces of pathological specimens of gastroscope biopsy or surgicalfrom nanchang university first affiliated hospital during January2007to September2009as the research objects, among which there are36pieces of ChronicNon-Atrophic Gastritis, CNAG,36pieces Chronic metaphastic atrophy gastritis,CMAG,38pieces of Dysplasia, Dys and39pieces of Gastric cancer, GC.Then testthe expression of FoxO3a and pFoxO3a transcription factors by immunohist-ochemical PV-9000method.2. The part of in-vitro part（1）Observe the change of morpholog on GES-1cell after Hp deal withdiffferent concentrations.（2）Select MTT method testing the influence of survival rate after Hp deal withGES-1cells by100:1,50:1,25:1.（3）Select Western-blot method testing the change of FoxO3a、pFoxO3a、p27and CyclinD1on protein expression level after Hp deal with GES-1cells by300:1,150:1,100:1.（4）Select real-time PCR method testing the change of FoxO3a、Bim and p27on mRNA expression level after Hp deal with GES-1cells by300:1,150:1,100:1.Research Findings：1The part of reasearch in-vivo（1） After analyse the H.pylori-positive gastric mucosal lesions with CNAG orMA, DYS or GC, we find there was no difference in FoxO3a expression among them（P＝0.183>0.05）.In the H.pylori-negative gastric mucosal lesions with CNAG orMA, DYS or GC, we find there is Statistical differences（P＝0.018<0.05）.It shows that a trend for reduced expression of FoxO3a in gastric mucosal lesions.And in thesame lesions in the stage FoxO3a’s expression in Hp negative group is higher than Hppositive group.During contrast we find that the FoxO3a’s expression of FoxO3a hasStatistical differences（P<0.05）， it shows that FoxO3a appears high in Hp negativegroup and low in Hp positive group.（2） After analyse the H.pylori-positive gastric mucosal lesions with CNAG orMA, DYS or GC, we find that pFoxO3a expression has no difference during them（P＝088>0.05）.In the H.pylori-negative gastric mucosal lesions with CNAG or MA,DYS or GC, we find there is statistical differences（P＝0.008<0.05）.It shows thatFoxO3a appear reduced trend in the early stage,but promote in the stage of gastriccancer.Analyse the expression of pFoxO3a.It shows that there is no statisticaldifferences（P<0.05）between the Hp negative group and Hp positive group.2. The part of reasearch in-vitro(1) We can find the typical morphological change of apoptosis by using invertedmicroscopy after Hp viable bacteria on GES-1cell. The contour of cell graduallybecome blurred, and shape become long,and form bubble.This will be more obviouslyas the more Hp and longer time.(2) MTT assay detect the affect of Hp on GES-1cell viability. We found thatGES-1cell viability is affected with the different concentration ratio of HP onGES-1,there is a significant difference (P <0.05). There is not all statisticallysignificant in the effect of Hp on cell viability. In the experiment of Time-anddose-dependent effects of Hp on GES-1cell viability, the survival rate of group100:1was significantly higher than that of group50:1and25:1. the survival rate of100:1was significantly higher than that of50:1and25:1(P=0.004<0.05)at0h. Thesurvival rate also decreased with the reduction of action concentration, and cellviability increased slightly at12h with statistical significance (P=0.035<0.05).Withthe extension of the duration of action, survival rapidly decreased. When the reactiontime reached at48h,100:1group survival rate was significantly lower than that of50:1and25:1groups, and there is significant difference (P=0.000<0.05), and therest of the time points no significant differences between each concentration, with nosignificant difference (P>0.05). (3) The effect of Hp on FoxO3a, pFoxO3a, p27and CyclinD1protein expressionwere observed by Wester blot. We observed that there is a difference in FoxO3aexpression in which the Ges-1cells were treated by different dose of H. pylori atdifferent time. An increasing trend is beginning to show at the point of0.5h, but thedownward trend come out after a period of peak(P <0.05),.The FoxO3a expressionpeak in150:1group(3h) presented earlier than that of100:1(6h).And it is not difficultto find FoxO3a expression was significantly lower at24h group than0h group.Therewere no significant differences in300:1group (P>0.05). the expression level ofpFoxO3a in the three concentration groups showed no significant difference (P>0.05).P27expression in the100:1and150:1showed significant difference (P <0.05), withFoxO3a. Cyclin D1expression of100:1and150:1showed significant difference,beginning to increase at0.5h, increasing gradually later, difficult to detect at24h. thatis basically the same at300:1group of former6h, but at the12h, expression beganto decline,and itis difficult to detectat24h.(4)The expression of FoxO3a mRNA has difference during three concentrationby real-time PCR, and showed significant difference (P <0.05).On100:1and150:1,the expression of FoxO3a mRNA has the trend come out then downward as thetime goes,and showed significant difference (P <0.05). There is no significantdifference (P>0.05) during the group of300:1.The expression of p27mRNA hassignificant difference (P <0.05) during three concentration.just in the group of150:1,shows the trend of come out then downward.There is no significant difference(P <0.05) in the group100:1and300:1,and no trend. This result is not fit to the resultof western blot.but we all know that the adjustment of the gene has adujstment aftertranscription, or we can say the transcription and translation level is not necessarilythe same.Because there exists decorate, transportation, degradation of mRNA andother small molecular weight mRNA, just like the regulate of miRNA.So theexpression of mRNA and protein of a molecular does not necessarily were positivelycorrelated.Conclusions:1、FoxO3a transcription factors involved in the happening and the developmentprocess of gastric mucosa related diseases caused by Hp, and play an important role in the early pathogenic during the infection of Hp.2、Different role of different concentrations of Hp in the normal gastric epithelialcells. The low concentration of Hp in the early stage can play a pro-apoptotic roleand play a pro-proliferation role at late stage. But the high concentration of Hp in theearly stage plays a pro-apoptotic role,which has no significant effect with time.3、Expression of FoxO3a and p27protein in normal gastric epithelial cells isbasically the same, first increased and then decreased,only slightly different inthe change time.Along with the prolongation of time, the expression ofcyclinD1show a downward trend, expression of Bim without significant changes,but in the long role of Hp,expression declined.At different period of Hp infectionmodel,FoxO3a produce a corresponding change in the regulation of its downstreamobjects target genes and plays role in Hp pathogenesis.