胃癌腹膜转移中PRL-3的分子调控机制研究

Phosphatase of regenerating liver-3 (PRL-3), a novel class of a superfamily of protein tyrosine phosphatases, is believed to be associated with cell motility, invasion, and metastasis. Our previous work found that PRL-3 is highly overexpressed in gastric cancer tissue with peritoneal metastasis and directly involved in the pathogenesis of gastric cancer peritoneal metastasis. Moreover, we further found that the down-regulation of endogenous miR-495 and miR-551a expression plays a causative role in overexpression of PRL-3 protein in gastric cancer peritoneal metastasis. However, the molecular regulation mechanisms by which endogenous miR-495/-551a expression is down-regulated and PRL-3 promotes gastric cancer peritoneal metastasis remain to be clearly elucidated. Therefore, in the present study, based on our previous findings, we will futher analyse whether DNA methylation is a major cause of the down-expression of endogenous miR-495/-551a, which results in PRL-3 overexpression in gastric cancer peritoneal metastasis by using a variety of molecular biology methods from aspect of gene promoter methylation inactivation. Moreover, this study will further explore the molecular regulation mechanisms by which PRL-3 promotes gastric cancer peritoneal metastasis from PTEN/PI3K/Akt signaling pathways and the change of MMP-2 and MMP-9 expression at the cellular and whole-animal levels. This study will provide new strategies for prevention and treatment of gastric cancer peritoneal metastasis.

肝再生磷酸酶3（PRL-3）属于蛋白酪氨酸磷酸酶超家族，与肿瘤的侵袭转移密切相关。我们在前一基金的资助下证实，PRL-3在胃癌组织中高表达并参与了胃癌腹膜转移，同时证实内源性microRNA分子miR-495及miR-551a表达低下是导致胃癌腹膜转移中PRL-3蛋白表达上调的重要原因。本研究拟在前期工作的基础上，从基因启动子甲基化表观遗传调控的角度，通过分子生物学手段进一步阐明胃癌腹膜转移中miR-495/-551a表达下调的分子调控机制；同时，在细胞和整体水平上，通过体外侵袭实验模型和胃癌裸鼠腹膜转移模型，从PTEN/PI3K/Akt信号通路及细胞内MMP-2、MMP-9表达改变入手深入全面地揭示PRL-3作为磷酸酶其作用的底物、调节的信号通路以及促胃癌腹膜转移的分子调控机制。该研究的顺利完成将有利于进一步揭示PRL-3的功能特性，也将为胃癌腹膜种植转移的防治提供新思路和新的干预环节。

肝再生磷酸酶3（PRL-3）属于蛋白酪氨酸磷酸酶超家族，具有一定的磷酸酶活性，在许多肿瘤的发生、发展中起重要作用。我们前期工作证实，PRL-3在胃癌组织中高表达并参与了胃癌腹膜转移，同时证实内源性microRNA分子miR-495及miR-551a表达低下是导致PRL-3蛋白表达上调的重要原因。然而，在胃癌腹膜转移过程中miR-495及miR-551a表达下调的调控机制以及PRL-3作为磷酸酶参与胃癌腹膜转移的分子机制尚未完全阐明。在本研究，我们采用甲基化特异性PCR（MSP）及亚硫酸氢钠测序法（BGS）等方法，同时借助甲基化抑制剂5-氮杂-2'-脱氧胞苷（5-Aza-dC），在细胞和组织水平，证实胃癌细胞中靶向调控PRL-3的miR-495表达下调是其基因启动子高甲基化表观遗传调控所致，而miR-551a表达低下与甲基化调控无关；此外，利用亲本的SGC7901胃癌细胞、野生型PRL-3高表达的SGC7901胃癌细胞、无磷酸酶活性的D72A/C104S突变型PRL-3高表达的SGC7901胃癌细胞以及PRL-3沉默的SGC7901胃癌细胞，同时结合PTEN-shRNA表达载体、PI3K抑制剂LY294002、Akt抑制剂API-2及MMP-2/9 inhibitor I，通过体外侵袭实验模型和胃癌裸鼠腹膜转移模型，我们进一步明确了PRL-3促胃癌腹膜转移的分子调控机制，即PRL-3通过其磷酸酶活性负调控PTEN，进而间接激活PI3K/Akt/NF-κB信号通路，上调MMP-2、MMP-9的表达，促胃癌腹膜转移。本项目进一步阐明了胃癌腹膜转移中PRL-3的功能特性，也为胃癌腹膜种植转移的防治提供了新思路和新的干预环节，具有一定的科学意义和应用价值。本课题全面完成研究计划，发表SCI论文5篇，中文核心期刊论文6篇，并完整培养硕士研究生4名。