PINK1-AS-miR-200a-Gαi1通路在胃癌中表达及功能研究

Molecularly-targeted therapy is the focus of clinical treatment and basic research for gastric cancer. Gαi1 is a novel and key signal molecule in receptor tyrosine kinases (RTKs) signaling. Our pre-experimental results show that Gαi1 expression was significantly up-regulated in human gastric cancer tissues, where miR-200a (the anti-Gαi1 miRNA) was down-regulated. In the cultured human gastric cancer cells, targeted-intervention of miR-200a-Gαi1 pathway significantly inhibited proliferation and migration of cancer cells. We further indentified lncRNA-PINK1-AS’s specific binding and inhibition of miR-200a. PINK1-AS is over-expressed in human gastric cancer tissues. We propose that PINK1-AS inhibits miR-200a to cause Gαi1 over-expression. The latter transduces RTK signaling and activates the downstream pathways, promoting gastric cancer cell progression. In the current proposal, we will exam the expression of PINK1-AS-miR-200a-Gαi1 pathway molecule in human gastric cancers, and their association with patients’ clinical manifestations. Using in vitro and in vivo models, the function and the underlying signaling mechanism of PINK1-AS-miR-200a-Gαi1-mediated gastric cancer cell progression will be studied. The results of the proposal will provide new molecular targets for clinical diagnosis and treatment of human gastric cancer.

分子靶向治疗是胃癌临床基础研究热点。Gαi1是新的受体酪氨酸激酶（RTKs）关键信号分子。预实验发现人胃癌组织中Gαi1表达显著上调，而miR-200a（Gαi1靶miRNA）下调。人胃癌细胞中调控miR-200a-Gαi1通路显著抑制癌细胞增殖、迁移。我们还发现lncRNA-PINK1-AS特异性结合并下调miR-200a。PINK1-AS在人胃癌组织中高表达。我们提出PINK1-AS竞争性结合抑制miR-200a，致Gαi1过表达，后者耦联RTKs激活下游信号并促胃癌细胞进展。本项目中，我们将检测人胃癌组织中PINK1-AS-miR-200a-Gαi1通路分子表达水平，分析它们与胃癌临床指标相关性；离、在体研究PINK1-AS-miR-200a-Gαi1通路在胃癌细胞进展中的作用和分子机制；并通过调控该通路达到抑制胃癌细胞生长的目的。为胃癌临床诊断和治疗突破提供新靶点。

胃癌是严重威胁人类健康的恶性肿瘤。胃癌的分子靶向治疗是研究热点。我们的前期研究证明，Gαi1对受体酪氨酸激酶(RTK)信号传导至关重要。本项目中，我们检测了Gαi1的促胃癌作用，我们发现 Gαi1 在人胃癌组织中上调，且与较差的总体存活率相关。在人胃癌细胞株和原代胃癌细胞中，Gαi1 shRNA沉默或敲除能显着抑制胃癌细胞活性，抑制细胞增殖和迁移，并诱导细胞凋亡。相反，Gαi1过表达促进体外胃癌细胞的增殖和迁移。通过检测肿瘤抑制性miRNA，microRNA-200a（miR-200a），我们发现miR-200a直接沉默Gαi1表达从而抑制胃癌细胞活性。人胃癌组织中 miR-200a 的表达下调，而靶向miR-200a的长链非编码RNA，PINK1-AS表达上调。RNA免疫沉淀、RNA-pulldown和RNA荧光原位杂交测定证实PINK1-AS直接与miR-200a结合。在胃癌细胞中沉默PINK1-AS导致miR-200a表达上调、Gαi1表达下调并抑制体外胃癌细胞进展，而PINK1-AS上调则产生相反的结果。进一步实验证明，由PINK1-AS shRNA诱导的抗胃癌细胞活性行为因miR-200a抑制或Gαi1的过表达而得到缓解。体内实验中，PINK1-AS shRNA抑制裸鼠皮下MGC-803移植瘤的生长，但PINK1-AS过表达则加速移植瘤的生长。原代胃癌细胞移植瘤内注射 PINK1-AS shRNA 慢病毒显著抑制移植瘤在裸鼠中的生长。总之，PINK1-AS 通过竞争性结合miR-200a促进Gαi1驱动的胃癌进展。靶向PINK1-AS-miR-200a-Gαi1轴对胃癌的临床诊断预后和治疗可能具有重要的价值。