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。Hp生物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MLH1和MSH2水平的显著降低，以及一个报告基因的微卫星不稳定性（MSI）型突变。高达30%的GC显示msi -高，慢性胃炎和肠化生的胃黏膜常显示msi -突变，msi阳性的GC患者更容易发生活动性Hp胃炎。这些数据支持我们的假设，即Hp可能通过损害DNA MMR导致胃上皮细胞的突变积累，这代表了胃癌发展的途径，并至少部分解释了Hp感染增加胃癌风险的原因。具体目的一：确定MSI突变积累所需的MMR缺乏程度，表征突变靶点和突变的谱，并确定Hp诱导培养胃上皮细胞（GEC）中MLH1和MSH2蛋白水平降低的基本机制。GEC和越来越多的Hp将共同培养。与MSI发展相关的MLH1和MSH2蛋白水平将被确定。使用GFP报告载体，通过western和FACS分析，确定重复暴露于Hp生物体的GEC中MSI突变积累。我们将确定polyCA和polyA重复序列的突变谱。Hp感染后检测MSH2和mlh1的转录率、mRNA和蛋白稳定性。具体目的二：研究人类幽门螺杆菌胃炎期间MLH1和MSH2基因的改变以及MSI突变积累的水平和频率。将通过激光捕获显微解剖，从Hp感染者和对照组非感染患者的Hp根除前后的活检中获得胃上皮。western和Taqman分析检测MLH1和MSH2蛋白和mRNA水平。Hp感染个体的上皮突变将通过检查推荐的微卫星标记和msi诱变的基因靶点来评估。在Hp根除前后检测MSI、MLH1和MSH2蛋白及mRNA水平，以检测Hp诱导的改变是否可逆。本研究的长期目标和影响是了解导致胃癌风险增加的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慢性Hp感染患者监测和GC风险评估的分子工具。从这项研究中获得的知识可能支持幽门螺杆菌感染患者根除Hp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.