人群为基础的胃癌及癌前病变血浆长链非编码RNA标志物研究

Gastric cancer (GC) is an end result of multifactorial and multistep process. Helicobacter pylori (H. pylori) infection plays a crucial role in gastric carcinogenesis. Long non-coding RNAs (lncRNAs) in human plasma have shown great potential as powerful and non-invasive cancer biomarkers. Study on lncRNAs as putative biomarkers of gastric cancer and its precursors may add to further understanding of the diseases. The results may also be useful for GC prevention and early diagnosis. Based on a population-based upper gastrointestinal cancer screening program, we aim to examine the profile of specific lncRNAs biomarkers for GC and its precursors using a multistage nested case-control design in a total of 820 subjects. By using the Arraystar Human LncRNA Microarray V4.0, a total of 40916 lncRNAs and 34235 mRNAs would be detected in 10 GC cases and 10 normal controls. Specific lncRNAs profile which might be aberrantly expressed in GC would be identified and further validated by quantitative Real-Time PCR method. The results will also be evaluated in two independent case-control studies including a training dataset and a validation dataset. Specific lncRNAs markers for GC would be further evaluated in GC precursors including severe dysplasia, mild dysplasia and intestinal metaplasia. We would also analyze the relationship between lncRNAs expression and H. pylori infection on gastric carcinogenesis. The functional mechanisms of validated lncRNAs markers of GC and its precursors would be investigated using bioinformatics.

胃癌发生是多因素参与的多步骤过程,幽门螺杆菌是胃癌发生的明确致病因子。血浆lncRNAs是有望成为胃癌及癌前病变早期诊断标志物的重要分子。本研究以参与“上消化道癌筛查的前瞻性评价研究”队列人群为基础，采用巢式病例-对照研究设计，首先在10例早期胃癌患者和10例正常者血浆样本中采用高通量人类长链非编码RNA芯片，检测40916种lncRNAs和34235种mRNAs在人体血浆的表达水平，初步筛选出差异表达的lncRNAs。在此基础上进一步扩大样本量，通过多阶段验证方式在合计820例样本中采用real-time PCR检测血浆lncRNAs分子在胃癌、重度异型增生、轻度异型增生、肠上皮化生和对照组的表达水平； 结合研究对象的幽门螺杆菌感染状况，探索lncRNAs作为胃癌及癌前病变早期标志物的可行性，并对相关lncRNAs初步开展生物信息学研究。

循环长链非编码RNA（lncRNA）是近年来非侵入性生物标志物研究中重要的一类。本研究采用巢式病例-对照研究设计，依托人群筛查项目中胃癌高发现场队列人群开展。利用高通量LncRNA芯片技术，及多阶段样本验证，筛选差异表达lncRNAs，探讨循环lncRNAs与幽门螺杆菌（H. pylori）感染及胃癌和胃重度不典型增生发病风险的关系，评价循环lncRNAs作为潜在胃癌标志物的价值。.本研究发现：.（1）高通量LncRNA芯片结果：通过对10例胃癌和10例非癌对照血浆样本进行高通量芯片检测（人类LncRNA芯片V4.0），共发现差异表达的1206个lncRNAs和728个mRNAs。浓缩筛选得到8个候选lncRNAs进入下一阶段验证。基因本体分析获得了可能与胃癌相关的GO条目60条，富集通路分析获得了胃癌相关18条通路。.（2）候选lncRNAs小样本验证：采用RT-qRCR方法在10例胃癌和10例对照中进行芯片结果验证。显示RP11-521D12.1，AC011995.3，RP11-5P4.3以及RP11-244K5.6在胃癌组的表达水平与对照组相比显著升高（P<0.05）。.（3）扩大样本验证：以胃癌/重度不典型增生作为病例组，正常/浅表性胃炎者为对照，对小样本验证发现的4个差异性候选lncRNAs，采用武威地区样本，在92例病例组和184例对照血浆中进行扩大样本验证，发现RP11-244K5.6在病例组中的表达水平高于对照组，差异具有统计学意义（P=0.038）。同时采用林州地区214例样本进行验证，结果同样证实RP11-244K5.6在病例组表达水平显著高于对照组（P=0.028）.（4）血浆RP11-244K5.6表达水平与胃癌/重度不典型增生的关系：采用多因素分析，调整年龄、性别、H. pylori感染和吸烟因素的关系对武威样本分析发现：与血浆中RP11-244K5.6低表达者相比，RP11-244K5.6高表达者发生胃重度不典型增生以上的风险显著升高（OR 2.67, 95% CI 1.14-6.25）。联合作用分析显示，与RP11-244K5.6低表达且H. pylori感染阴性者相比，RP11-244K5.6高表达且H. pylori阳性者发生胃癌的风险升高了2.51倍(OR 3.51, 95%CI 1.52-8.07)。