Skp2的磷酸化调控胃癌细胞自噬及肿瘤进展的机制研究

With the funding of one-year project of NSCF,we confirmed that the ubiquitination of Beclin 1 by Skp2 leads to the decrease of Beclin 1 expression in gastric cancer, and may induce the gastric cancer cell autophagy.In clinical specimens of gastric cancer, the expression of Skp2 and Beclin 1 significantly correlated. Moreover,we found that mTOR phosphorylate Skp2 in Ser64,which significantly enhance the ubiquitination of Beclin 1. In current project, by constructing Skp2 phosphorylation antibody, mutant Skp2 S64 and downgrading mTOR expression in gastric cell, we continue to detect autophagic molecular expression in vivo and in vitro.We will also show the regulation of autophagy by Skp2 is relying on the Beclin 1 ubiquitination and mTOR phosphorylation.Using the skp2 inhibitor, we try to target the autophagy pathway to treat gastric cancer. This study will clarify the molecular mechanism that the phosphorylation of Skp2 by mTOR mediate ubiquitination of Beclin 1 regulating gastric cancer cell autophagy and tumor progression.It may provide new train of thought of diagnosis and treatment of gastric cancer.

在去年国家自然基金一年期小额项目资助下，我们证实：Skp2泛素化降解Beclin 1导致胃癌中Beclin 1表达降低，并诱导胃癌细胞自噬；在临床标本中，Skp2与Beclin 1的表达显著相关。我们在实验过程中，进一步发现Beclin 1泛素化受mTOR磷酸化Skp2的影响。mTOR在Ser64位磷酸化Skp2后，Beclin 1的泛素化降解明显增强。本课题将合成Skp2磷酸化抗体，构建Skp2突变体并下调/过表达mTOR及Beclin 1，检测自噬通路相关蛋白表达，观测电镜下自噬泡变化，验证胃癌细胞中自噬的调节依赖mTOR磷酸化及Beclin1的泛素化，同时Beclin 1能逆转Skp2调控作用；通过临床标本检测该通路相关蛋白，在体内外实验中采用Skp2抑制剂探索抑制该通路靶向治疗胃癌可行性。本研究从磷酸化及泛素化双向调控细胞自噬的角度阐明胃癌进展机理，将为胃癌诊治提供全新的思路。

mTOR和Skp2都在胃癌的发生发展中起着重要的作用，然而，他们之间潜在的作用机制并不明确。本课题首次发现mTOR磷酸化Skp2后可以阻止Skp2的泛素化降解，磷酸化位点发生在64位丝氨酸，突变该位点，可以抑制胃癌的增殖转移，在细胞和动物实验上都得到了证明。具有重要的科学意义。我们同时证明了联合检测mTOR和Skp2 S64可以预测胃癌患者的预后，并提示联合应用mTOR抑制剂和Skp2抑制剂可以更有效的靶向治疗胃癌，具有重要的临床应用前景。