SLP2抑制PHB泛素化降解促进胃癌细胞增殖和转移的正反馈调控环路

SLP2 was previously screened as a potential oncogene and proved to be up-regulated and associated with poor prognosis in patients with gastric cancer. However, the certain mechanism of SLP2 in gastric cancer is still unclear. To address this issue, PHB was selected to potentially interact with SLP2 using a yeast two-hybrid system. Moreover, we noticed that degradation of PHB by ubiquitination and inhibition of MAPK signaling pathway was prompted in SLP2-silenced cells. In addition, the endogenous SLP2 was elevated when the exogenous SLP2 was transfected, which indicated a potential positive feedback loop of SLP2. Interestingly, we also found that there were binding sites of ELK1, as a downstream transcript factor of MAPK signaling pathway, in the promoter region of SLP2. Therefore, we propose the hypothesis of the positive feedback loop: SLP2 might activate the MAPK signaling pathway and further promote transcriptional activity of ELK1 through the inhibited degradation of PHB ubiquitination. We designed the following studies to verify the hypothesis: first, the interaction of SLP2 and PHB could decrease the degradation of PHB by ubiquitination. Second, the interaction of SLP2 and PHB could activate MAPK signaling pathway and then induce the activation of ELK1. Lastly, ELK1 was potential to stimulate the transcription of SLP2. The study will greatly promotes our recognition on the regulatory mechanism of SLP2 in the proliferation and metastasis of gastric cancer.

前期我们将SLP2作为候选癌基因发现其在胃癌组织中表达升高且与患者预后不良相关，但其调控机制尚不清楚。前期工作中，我们通过酵母双杂交筛选得到相互作用蛋白PHB，发现沉默SLP2可促进PHB泛素化降解并抑制MAPK信号通路激活。此外，在胃癌细胞中导入外源性SLP2可促进内源性SLP2的表达，提示SLP2可能存在自身正反馈调控。分析发现SLP2启动子区域存在MAPK信号通路下游分子ELK1的结合位点。由此我们提出一个正反馈调控环路的设想：SLP2可通过抑制PHB泛素化降解活化MAPK信号通路进而促进ELK1对SLP2的转录。为验证该设想，我们拟进一步证明：一、SLP2通过与PHB相互结合抑制其泛素化降解；二、SLP2与PHB相互作用活化MAPK信号通路，从而诱导ELK1激活；三、转录因子ELK1促进SLP2的转录。该项目的顺利完成有利于提高对SLP2参与胃癌细胞增殖和转移的重要调控机制的认识。

胃癌的发病率和死亡率高，在我们前期研究中，我们利用二维凝胶电泳筛选正常胃上皮粘膜组织和胃癌组织的差异表达蛋白，通过质谱分析得到 39 多个差异表达蛋白。通过免疫组化验证发现SLP2表达在胃癌组织中表达明显升高且与患者的预后不良相关。基于此，我们拟进一步探索SLP2在胃癌中的表达及其临床意义。在研究内容上，我们首先进一步扩大临床患者胃癌样本量分析SLP2的表达情况并分析其与患者病理学参数的关系。进一步我们利用细胞功能实验分析SLP2对胃癌细胞增殖和侵袭能力的影响。在机制上，我们拟进一步明确SLP2与PHB的相互结合情况，并确定两者的调控关系，同时分析SLP2对MAPK信号通路的影响。最后，我们拟明确转录因子ELK1对SLP2的转录激活作用。通过内部和外部临床样本和生存分析我们发现SLP2在胃癌组织中表达显著升高并提示患者预后不良，SLP2可与PHB直接集合，抑制E3泛素连接酶SKP2与PHB的结合，从而稳定PHB在细胞中的表达，进一步激活MAPK信号通路关键分子Raf1、MEK1/2，ERK1/2及下游分子ELK1的磷酸化。利用Crisper-cas9技术敲除SLP2的表达可显著抑制胃癌细胞的增殖和侵袭能力。在体内实验中我们也发现SLP2缺失可显著抑制小鼠皮下瘤的生长。我们通过数据库分析发现SLP2的下游分子ELK1潜在转录调控SLP2。通过染色质免疫共沉淀、EMSA和双荧光报告素酶系统检测，我们确定ELK1可与SLP2的转录启动区域结合，正向调控SLP2的转录。因此，我们验证了SLP2-PHB-MAPK-ELK1-SLP2 形成正反馈调控环路的设想。该项目的顺利完成，解释了胃癌中SLP2显著增高的原因，也发现SLP2与PHB结合在胃癌细胞中的重要作用，通过干扰SLP2与PHB的结合可能为胃癌治疗提供新的策略。