MTA2基因调控胃癌侵袭及转移的分子机制研究

Tumor invasion and metastasis are the important prognostic factors of gastric cancer, their related molecular mechanisms are the hotspots in both clinical and basic research. The preliminary results of our team revealed that the expression of metastatic associated 1 family member 2, MTA2, was related with the depth and lymph node status of gastric cancer, MTA2 knockdown might inibit the migration and invasion ability of gastric cancer cell lines SGC-7901 and AGS, concomitant with the downregulation of tumor invasion-related genes such as CD24, indicating that MTA2 might involve in the invasion and metastasis of gastric cancer. The aim of this study is to investigate the precise molecular mechanisms of MTA2 on the invasion and metastasis of gastric cancer, and its transcriptional regulation mechanism, so as to provide preclinical data in exploring the potential interventional targets in MTA2 related pathways. In detailed working schedule includes: (1) changing the MTA2 gene expression status in gastric cancer cell lines by knock-down or knock-in techniques, and observe the consequent changes of cellular biological behaviors;(2) the results of gene expression chip on the MTA2 knock-down cell line indicated that CD24 is the downstream gene of MTA2. In this study, the role of CD24 in MTA2 related gastric cancer cell invasion and metastasis will be investigated,and the transcriptional regulation of CD24 by MTA2 will also be explored;(3) the mechanism of transcriptional regulation of MTA2 in gastric cancer cells will be investigated by luciferase-report gene assay and ChIP technique, the relationship between targeted transcriptional factors and MTA2 will be further investigated in clinical specimens.

肿瘤侵袭及转移是影响胃癌患者预后的重要因素。本课题组前期发现MTA2基因与胃癌浸润深度、淋巴结转移等恶性表型密切相关，敲低MTA2表达能明显抑制胃癌细胞SGC-7901及AGS迁移及侵袭；并伴CD24等基因的表达下调。本研究拟阐明MTA2基因通过CD24影响胃癌细胞侵袭及转移的具体分子机制，并明确MTA2在胃癌表达的转录调控机制；为探索以MTA2相关通路为靶点的分子靶向治疗奠定基础。具体内容包括：1）调节胃癌细胞MTA2表达状态，观察转移相关生物学行为变化，结合临床标本检测，明确其在胃癌侵袭转移中的作用；2）明确CD24在MTA2调控的胃癌细胞侵袭及转移中的作用机制，并探索MTA2是否参与CD24转录调控；3）通过荧光素酶报告基因、染色质免疫共沉淀等技术，结合临床标本检测，明确MTA2基因在胃癌表达的转录调控机制。

本课题从组织、细胞、分子三个层次，系统地研究了转移相关基因2（metastasis associated 1 family member 2，MTA2）在胃癌中的生物学功能，及其上下游调控机制。结果显示，下调MTA2基因表达（shRNA转染）可明显抑制胃癌细胞SGC-7901及AGS软琼脂克隆形成、划痕愈合、迁移及侵袭能力，上调MTA2基因表达（MTA2表达质粒）则增强胃癌细胞BGC-823及MKN28克隆形成能力。体内试验中，下调MTA2明显抑制SGC-7901胃癌细胞皮下成瘤及肺转移能力，反之，上调MTA2显著增强BGC-823胃癌细胞皮下移植瘤及肺转移灶生长能力。. 通过表达基因芯片分析MTA2上调及下调后BGC-823及SGC-7901胃癌细胞内差异表达基因，显示12个基因为两组数据共有且具有显著倍率改变。IL-11表达倍率在SGC-7901/shMTA2细胞中为0.2631（P=0.0054），在BGC-823/MTA2细胞中为7.3145（P=7.74e-06）。外源性给予重组人IL-11可恢复SGC-7901/shMTA2细胞体外克隆形成能力（克隆形成数：SGC-7901/shMTA2/rhIL-11比SGC-7901/shMTA2/PBS：25.6 ± 1.5比20.0 ± 2.3，P=0.015）。显示IL-11是MTA2调控胃癌细胞生长的下游效应分子。. 进一步分析MTA2在胃癌中表达的转录调控机制。发现MTA2启动子关键调控区域位于其转录起始密码子上游-482bp至-323bp处。转录因子Sp1与MTA2在胃癌中表达呈正相关，上调Sp1表达可增强MTA2启动子转录活性，但下调Sp1未见MTA2表达抑制。在MTA2启动子关键转录调控区域发现转录因子TFAp2A结合位点。下调SGC-7901及MKN45胃癌细胞中转录因子TFAp2A（shRNA转染）可显著抑制MTA2表达及其启动子转录活性，并明显抑制SGC-7901及MKN45胃癌细胞克隆形成能力及IL-11 mRNA表达。组蛋白去乙酰化酶抑制剂SAHA可下调TFAp2A、MTA2及IL-11表达，并下调MTA2启动子转录活性。以上结果显示，胃癌中存在TFAp2A/MTA2/IL-11通路，参与调控胃癌细胞生长。MTA2表达还与细胞组蛋白乙酰化水平相关。