肿瘤微环境中基质细胞高表达MLCK促进胃癌转移的机制研究

Accumulating evidence has indicated that cancer progression is regulated by interactions between tumor cells and activated stromal cells. Our previous study found that MLCK was highly expressed on stromal cells of gastric cancer, and correlated with tumor stage and active phenotypes of fibroblasts. In the present study, we aim to remodel fibroblast-dependent tumor microenvironment and determine the role of MLCK in the metastasis of gastric cancer. Aim 1: To determine the relationship between MLCK expression in stromal cells and clinicopathological features of gastric cancer by analyzing the expression of MLCK in gastric cancer and stromal fibroblasts; Aim 2: To define the role of MLCK in the metastasis of gastric cancer in vitro and in vivo by downregualting of MLCK expression in CAF with RNAi or specific inhibitor; Aim 3: To study the mechanism of MLCK in the metastasis of gastric cancer by analyzing the differential expression of molecules in CAF with or without MLCK knockdown; Aim 4: To explore the molecular mechanism of MLCK regulation in CAF by tumor microenvironment in gastric cancer. Thus, our study identified the critical role of CAF-MLCK in cancer-associated inflammation, EMT of gastric cancer cells and active phenotype of CAF, which all favored the metastasis of gastric cancer, at various levels from gene expression, protein regulation to system biology. These findings should thereby ultimately contribute to the development of novel CAF-targeting therapies for the metastasis of gastric cancer.

研究发现微环境中肿瘤细胞和基质细胞的相互作用对肿瘤进展发挥着重要作用。本课题组前期工作发现MLCK高表达于胃癌基质中，其表达量与胃癌临床分期和成纤维细胞活化等密切相关。因此我们通过模拟建立成纤维细胞依赖的胃癌微环境模型，探讨MLCK在胃癌转移中的作用及机制：1）首先分析MLCK在胃癌组织和基质成纤维细胞中表达，揭示MLCK与胃癌转移的相关性；2）通过RNAi或化学抑制剂下调CAF中MLCK表达和活性，阐明MLCK表达对胃癌细胞体内外转移能力的影响；3）通过高通量芯片筛查CAF中MLCK下调前后差异分子，探讨MLCK影响胃癌转移的分子机制；4）探讨胃癌微环境调控CAF中MLCK表达的分子机制。本课题以MLCK为切入点，从基因转录差异、蛋白表达调控以及系统生物学的层面，阐明它在胃癌微环境中炎症反应、胃癌细胞EMT及成纤维细胞活化相关的分子机制，为临床干预胃癌转移提供新的理论与实验依据。

肿瘤微环境在肿瘤进展中的作用逐渐受到关注，肿瘤细胞与邻近基质细胞的相互作用形成利于肿瘤生长的微环境，促进了肿瘤的侵袭和转移。肿瘤相关成纤维细胞是微环境中最主要的一种基质细胞，在肿瘤的进展中发挥着重要作用，但其调控和作用机制仍未完全阐明。本课题组研究工作发现：1）肌球蛋白轻链激酶MLCK高表达于胃癌基质CAFs中，其表达量与胃癌临床分期和成纤维细胞活化等密切相关，然而胃癌细胞中MLCK基因呈现高度甲基化并可以作为胃癌的分子预警标志物。2）为进一步分析高表达MLCK的CAFs在胃癌侵袭转移的作用机制，我们成功分离了胃癌相关成纤维细胞，发现与癌旁正常成纤维细胞相比，CAFs具有较强的促肿瘤体内生长能力。通过siRNA下调CAFs中MLCK表达，抑制其促胃癌细胞体外增殖、迁移、侵袭能力和体内生长能力。3）我们通过共培养体系探讨了CAFs与胃癌细胞的“对话”机制，发现HGF是CAFs分泌的一个重要分子，通过旁分泌途径促进邻近基质正常成纤维细胞活化和胃癌细胞的体内外增殖、迁移，通过HGF中和抗体或siRNA中和或下调CAFs中HGF的表达后，可以抑制胃癌的进展。4）通过 MLCK抑制剂特异性下调CAFs 中 MLCK 的活性后，抑制了HGF诱导的CAFs活化。5）我们通过cDNA芯片初步分析了MLCK下调前后CAFs中基因的差异表达，并通过GO分析发现MLCK下调后，CAFs中主要与pathway in cancer、focal adhesion、JAK-STAT signaling pathway等相关的分子发生明显改变，表明MLCK主要调控了CAFs的肿瘤生物学相关特征。本课题以MLCK为切入点，从基因转录差异、蛋白表达调控以及系统生物学的层面，阐明CAFs在胃癌进展中的分子机制，为临床干预胃癌转移提供新的理论与实验依据。