MMP-2/9在胃癌患者胃液芳香族氨基酸含量变化中的作用机制

In the previous study, we found that the level of aromatic amino acids (AAAs) in gastric juice from gastric cancer patients was higher than non-neoplastic gastric disease patients, which is expected to become biomarkers for early detection of gastric cancer, but the underlying mechanism of this phenomenon is still not clear. In this study, we will clarify the role of proteolytic enzymes MMP-2 and MMP-9 in the difference of gastric juice AAAs content between gastric cancer, gastric dysplasia and chronic gastritis patients from extracellular metabolism primarily. Therefore, we put forward the scientific hypothesis that treating type IV collagen as target, MMP-2/9 as driving factor, AAAs as metabolic products. Specific steps are as follows: To begin with, the correlation between MMP-2/9 and AAAs was clearly defined at human tissue and cell level respectively. Secondly, it was proved that through knockdown or overexpression of MMP-2/9 gene in human gastric carcinoma cells, type IV collagen could be degraded more or less, result in the content of AAAs increase or decrease correspondingly. Finally, nude mouse transplantation tumor experiment was carried out to observe anti-tumor effects, initiating or promoting tumorigenesis and the change of AAAs content in transplanted tumor tissue of the mouse from RNA interference and gene overexpression. Above all, the ultimate goals of this project are to open a new approach for early diagnosis and screening of gastric malignancies, and to provide a theoretical basis for its clinical application.

课题组前期研究发现胃癌患者胃液芳香族氨基酸（AAAs）含量异常升高，有望成为胃癌早期诊断的生物标志物，但机制未明。本研究拟从细胞外代谢入手，以IV型胶原为靶点，MMP-2/9为切入点，AAAs为代谢产物，解释上述现象出现的原因。具体步骤如下：首先，分别在人体组织和细胞层面明确MMP-2/9与AAAs间的相关性；然后，通过细胞干预实验证明人胃癌细胞可以通过MMP-2/9降解IV型胶原产生AAAs；最后，通过动物实验证实MMP-2/9对裸鼠皮下种植瘤组织生长与AAAs含量的影响。旨在为胃癌的早期诊断和筛查提供新方法和新思路，为胃液AAAs含量检测临床应用提供理论依据。

代谢重塑与肿瘤的发生发展密切相关，肿瘤微环境内代谢物与肿瘤细胞的相互作用共同推动了疾病的进展。在课题组前期研究中，我们发现胃癌患者胃液内芳香族氨基酸（苯丙氨酸、酪氨酸和色氨酸）含量升高，并且具有潜在的诊断价值，但其升高的机制及其对肿瘤细胞的影响尚不明确。本课题从细胞外代谢入手，以基质金属蛋白酶MMP-2/9作为研究切入点，从组织学、细胞模型和动物模型三个方面探讨MMP-2/9是否是引起芳香族氨基酸含量升高的原因。研究结果发现， MMP-2/9在胃黏膜从慢性胃炎进展至胃癌阶段其表达水平逐渐升高并伴随细胞外基质内IV型胶原含量的减少，胃癌组织内MMP-2/9的高表达与胃癌患者胃液内芳香族氨基酸含量的升高具有正相关性，而IV型胶原含量的减少与芳香族氨基酸含量的升高呈负相关。基于胃液芳香族氨基酸含量的变化、胃液pH等指标我们构建了一胃液指数模型可用于胃癌的早期诊断。我们也进一步探究了基质金属酶家族其他成员在胃癌无创早诊中的潜在价值。进一步地，体外细胞模型研究发现肿瘤乏营养微环境可以促进胃癌细胞内MMP-2/9的表达上调和活性升高，进而可降解细胞外基质中的成分包括IV型胶原。但是，微环境内芳香族氨基酸含量的变化并不能影响胃癌细胞MMP-2/9的表达和其酶活性。在此基础上，我们运用靶向和非靶向代谢组学技术探究胃癌患者胃液内代谢物轮廓，发现除了氨基酸含量变化外，与三羧酸循环、糖酵解、嘌呤嘧啶核苷酸代谢相关的代谢物也在早期胃癌和进展期胃癌患者胃液内升高，并且具有潜在的诊断意义。此外，在课题研究进展中还针对非必须氨基酸-脯氨酸进行了拓展研究，探讨了胞外脯氨酸对胃癌细胞生物学行为的影响。以上研究结果为进一步揭示细胞外代谢调控异常与胃癌的发生发展提供了实验依据，研究发现的新的代谢物有可能作为胃癌早诊的潜在生物标志物。