H.pylori Effects on DNA Repair in Gastric Epithelium

描述（由申请人提供）：H.幽门螺杆菌（Hp）增加胃癌（GC）的风险是非常未知的。幽门螺杆菌生物体与胃上皮细胞共培养的直接相互作用导致主要DNA错配修复（MMR）蛋白MLH 1和MSH 2的水平显着降低，以及报告基因中的微卫星不稳定性（MSI）型突变。高达30%的胃癌表现为MSI-高，慢性胃炎和肠上皮化生的胃粘膜经常表现为MSI-突变，MSI阳性的胃癌患者更可能患有活动性Hp胃炎。这些数据导致我们的假设，幽门螺杆菌可能会导致突变积累在胃上皮细胞通过损害DNA MMR，代表了GC的发展途径，并解释至少部分如何幽门螺杆菌感染增加GC的风险。具体目标一：确定MSI突变累积所需的MMR缺陷程度，表征突变靶点和突变谱，并确定培养的胃上皮细胞（GEC）中Hp诱导MLH 1和MSH 2蛋白水平降低的基本机制。将GEC和增加数量的Hp共培养。将测定与MSI发展相关的MLH 1和MSH 2蛋白水平。将使用GFP报告载体通过Western和FACS分析在重复暴露于Hp生物体的GEC中确定MSI突变累积。我们将确定polyCA和polyA重复序列的突变谱。检测感染Hp后MSH 2和MLH 1的转录率、mRNA和蛋白质的稳定性。具体目的二：研究人肝癌细胞中MLH 1和MSH 2的变化，以及MSI突变积累的水平和频率。人类幽门螺杆菌性胃炎。胃上皮将通过激光捕获显微切割获得，使用Hp根除前后Hp感染个体和对照非感染患者的活检组织。将通过蛋白质印迹和Taqman分析测定MLH 1和MSH 2的蛋白质和mRNA水平。将通过检查推荐的一组微卫星标记和MSI诱变的基因靶点来评价Hp感染个体上皮中的突变。将在Hp根除前后评价MSI和MLH 1和MSH 2蛋白和mRNA水平，以测试Hp诱导的变化是否可逆。本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。表征MMR改变和靶基因突变可能成为有用的分子工具，监测和GC风险评估慢性Hp感染患者。从这项研究中获得的知识可能支持在H.幽门螺杆菌感染的患者，以防止GC。

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.