Sirt1通过去乙酰化修饰对胃癌细胞中STAT3调控功能的机制研究

Constitutive activation of STAT3 is important to maintain the malignant behavior of gastric cancer. Acetylation of STAT3 has recently been shown to maintain the stability of phosphorylated STAT3 and enhance the transcriptional activity. Sirt1, the deacetylase of STAT3, could decrease the stability of phosphorylated STAT3 and inhibit its transcripitonal activity. However, the function of Sirt1 has not been well understood in gastric cancer. Our previous study has revealed that aberrant activated STAT3 play an important role in drug resistance of gastric cancer cells, the further exploration found that Sirt1 may be involved in reduced proliferation of gastric cancer cells through inhibition of activated STAT3. Based on the previous observation,the hypothesis is that the transcriptional activity of STAT3 is regulated by Sirt1 in gastric cancer. To verify the hypothesis,we would employ molecular biological techniques as mentioned below. The expressions of Sirt1 and STAT3 would be examined and analysed in patients' gastric cancer tissues and cell lines. STAT3 site-directed mutants, in which loss of fucntion mutations were induced in acetylation sites or phosphorylation sites, would be constructed to explore the effects of these sites on the transcriptional activity of STAT3, while Sirt1 ShRNA lentivirus would be employed to study its function in gastric cancer cells. Further more, doxycyclin-induced Sirt1 knockdown mice would be established to study the functions and regulations of Sirt1 in vivo. This project would delineate the mechanisms of acetylation and phosphorylation of STAT3 in the tumorigensesis and development of gastric cancers, which was regulted by Sirt1 deacetylase. It would provide a potential approach in the treatment of gastic cancers.

胃癌中STAT3的持续激活对维持恶性表型具有重要作用，近来研究发现乙酰化是维持STAT3磷酸化激活的重要修饰方式。Sirt1通过STAT3去乙酰化降低其磷酸化水平从而抑制STAT3的转录活性；且Sirt1在胃癌中的功能尚存争议，其确切机制尚未阐明。我们的前期研究发现STAT3持续磷酸化激活参与了胃癌细胞的耐药，初步研究发现Sirt1可能通过抑制STAT3的磷酸化降低胃癌细胞的增殖，因此我们拟通过本课题验证这一假说。我们将在各期胃癌组织和细胞株中检测Sirt1与STAT3的表达水平，并用STAT3基因定点突变技术研究STAT3乙酰化和磷酸化对自身转录活性的影响，用ShRNA技术研究Sirt1对STAT3的功能调节。此外，我们还将建立药物诱导型Sirt1基因敲除的小鼠胃癌模型，在体内观察其功能及相关机制。本课题将全面阐述Sirt1介导STAT3参与胃癌发生发展的分子机制，为胃癌防治提供新策略。

胃癌中 STAT3 的持续激活对维持恶性表型具有重要作用，近来研究发现乙酰化是维持 STAT3 磷酸化激活的重要修饰方式。Sirt1 通过 STAT3 去乙酰化降低其磷酸化水平从而抑制 STAT3 的转录活性;且 Sirt1 在胃癌中的功能尚存争议，其确切机制尚未阐明。我们的前期研究发现 STAT3 持续磷酸化激活参与了胃癌细胞的耐药，初步研究发现 Sirt1 可能通过抑制 STAT3 的磷酸化降低胃癌细胞的增殖。我们在不同阶段胃癌组织（胃癌前病变、早癌、进展期胃癌）中，使用免疫组化方法检测Sirt1、STAT3及磷酸化STAT3水平，发现与正常胃粘膜相比，在不同阶段胃癌组织中Sirt1表达水平都是增高的，而同样，STAT3及磷酸化STAT3在不同阶段胃癌组织中保持相对高水平，预示着高水平的Sirt1表达可能是胃癌形成的因素之一。通过对这部分患者的随访发现，低表达SIRT1及低表达STAT3的患者生存时间比高表达的患者生存时间较长；而二者都低表达的情况下，患者生存时间最长。体外实验发现，SIRT1低表达后胃癌细胞的增殖能力增强，迁移及侵袭能力增强；而SIRT1过表达后胃癌细胞的克隆形成能力和迁移侵袭能力降低。此外，我们还发现干扰胃癌细胞SIRT1表达后，STAT3的在基因和蛋白水平的表达总量是基本稳定的，而STAT3的乙酰化和磷酸化水平是增加的；同样，过表达SIRT1之后乙酰化STAT3的水平是减少的。据此我们猜想Sirt1和STAT3可以成为胃癌早期诊断及预测预后的标志物，为胃癌防治提供新策略。并且本课题认为，Sirt1在人体中更多的是扮演“守门人”角色，即Sirt1在体内达到某一平衡水平，才会使胃粘膜处于正常状态，因此Sirt1更像是胃癌的保持基因，而不是胃癌的促成基因。