H.pylori Effects on DNA Repair in Gastric Epithelium

描述(由申请人提供)：幽门螺杆菌(Hp)增加胃癌(GC)风险的分子机制尚不清楚。幽门螺杆菌与胃上皮细胞共培养后，其主要DNA错配修复(MMR)蛋白MLH1和MSH2的水平显著降低，报告基因的微卫星不稳定性(MSI)型突变显著减少。高达30%的GC表现为MSI-High，慢性胃炎和肠化生的胃粘膜经常出现MSI突变，MSI阳性的GC患者更容易发生活动性Hp胃炎。这些数据导致了我们的假设，即Hp可能通过损害DNA MMR导致胃上皮细胞突变积累，这代表了GC发展的一条途径，并至少部分解释了Hp感染如何增加GC风险。具体目标一：确定MSI突变积累所需的MMR缺陷的程度，表征突变靶点和突变的谱，并确定Hp诱导培养的胃上皮细胞(GEC)MLH1和MSH2蛋白水平降低的基本机制。GEC和越来越多的HP将进行共培养。与MSI发生相关的MLH1和MSH2蛋白的水平将被确定。通过Western和FACS分析，将使用GFP报告载体确定GEC重复暴露于Hp生物中MSI突变的累积。我们将确定PolyCA和PolA重复序列的突变谱。检测Hp感染后MSH2和MLH1的转录速率、mRNA和蛋白质的稳定性。目的二：研究幽门螺杆菌胃炎时MLH1和MSH2的变化及MSI突变的水平和频率。胃上皮细胞将通过激光捕获显微切割获得，取自HP感染患者根除Hp前后和对照未感染患者的活检组织。MLH1和MSH2的蛋白和mRNA水平将通过Western和Taqman分析来确定。将通过检查一组推荐的微卫星标记和微卫星突变的基因靶标来评估HP感染者上皮细胞的突变。在根除Hp前后检测MSI、MLH1和MSH2蛋白和mRNA水平，以检验Hp引起的变化是否可逆。这项研究的长期目标和影响是了解导致GC风险增加的HP细菌-宿主相互作用机制。MMR改变和靶基因突变的特征可能成为监测和评估慢性Hp感染患者GC风险的分子工具。从这项研究中获得的知识可能支持根除幽门螺杆菌感染患者以预防GC的指征。

DESCRIPTION (provided by applicant): The molecular mechanisms by which H. pylori (Hp) increases gastric cancer (GC) risk are vastly unknown. Direct interaction of Hp organisms in co-culture with gastric epithelial cells causes a marked decrease in the levels of the main DNA mismatch repair (MMR) proteins MLH1 and MSH2 and microsatellite instability (MSI)-type mutations in a reporter gene. Up to 30% of GC show MSI-High, gastric mucosa with chronic gastritis and intestinal metaplasia frequently show MSI-mutations, and patients with MSI-positive GC are more likely to have active Hp gastritis. These data lead to our hypothesis that Hp might cause mutation accumulation in the stomach epithelium by impairing DNA MMR, representing a pathway of GC development and explaining at least in part how Hp infection increases GC risk. Specific aim one: To determine the degree of MMR deficiency required for MSI mutation accumulation, to characterize the spectrum of mutational targets and mutations and to identify the fundamental mechanisms underlying the reduced levels of MLH1 and MSH2 proteins induced by Hp in cultured gastric epithelial cells (GEC). GEC and increasing numbers of Hp will be co-cultured. The levels of MLH1 and MSH2 proteins associated with MSI development will be determined. MSI mutation accumulation will be determined in GEC repeatedly exposed to Hp organisms using GFP reporter vectors by western and FACS analyses. We will determine the spectrum of mutations at polyCA and polyA repeats. Transcription rates, mRNA and protein stability of MSH2 and MLH 1 will be measured after Hp infection. Specific aim two: To characterize the alterations of MLH1 and MSH2 and level and frequency of MSI mutation accumulation during H. pylori gastritis in humans. Gastric epithelium will be obtained by laser capture microdissection using biopsies from Hp infected individuals before and after Hp eradication and from control non-infected patients. Protein and mRNA levels of MLH1 and MSH2 will be determined by western and Taqman analysis. Mutations in the epithelium of Hp infected individuals will be evaluated by examining a recommended panel of microsatellite markers and gene targets of MSI-mutagenesis. The MSI and MLH1 and MSH2 protein and mRNA levels will be evaluated before and after Hp eradication to test whether the changes induced by Hp are reversible. The long-term goals and impact of this study are to understand the Hp bacterial-host interaction mechanisms that lead to increased risk of GC. Characterization of MMR alterations and target gene mutations may become useful as a molecular tool for surveillance and GC risk assessment of patients with chronic Hp infection. Knowledge gained from this study is likely to support the indication for Hp eradication in H. pylori-infected patients to prevent GC.