肝细胞癌转移相关磷酸化蛋白质的鉴定、调控及其作用机制的研究

Metastasis and recrudesce is the sticking point of hepatocellular carcinoma (HCC) research, but also to prolong survival and improve the long-term efficacy of bottlenecks.Protein phosphorylation with HCC recurrence and metastasis are closely related.The preliminary work of the Task through "phosphopeptide's enrichment by Ti4+-IMAC,steady isotope(dimethyl) labelling and detection of LC-MS/MS " this new phosphorylation proteomics quantitative analysis technology line, to analysis HCC tissue samples,have detected many phosphorylated protein such as p-PED/PEA-15(s-116),p-P27-T187 which the level and extend of phosphorylation can regule apoptosis,transformation and infestation of cell.On this basis, the phosphorylation proteomics quantitative analysis of the technical analyst with different metastatic potential of hepatocellular carcinoma cell line phosphorylated proteins expression profiling, high throughput screening of tumor metastasis-related phosphorylated proteins, phosphorylation sites kinase; further meaningful differences in phosphorylation of proteins downstream functional analysis, the use of point mutations and specific protein kinase inhibitor method to observe the modification of the dephosphorylation of its protein expression, intracellular distribution and transfer characteristics of hepatocellular carcinoma cells impact.Aimed at reveal the molecular mechanism of HCC recurrence and metastasis,looking for a better transfer of indicators of HCC prediction and intervention target molecules.

肝癌转移复发是攻克肝癌的关键，也是延长生存期、提高远期疗效的瓶颈问题。蛋白质磷酸化修饰与肝癌的转移复发密切相关。本课题组前期工作中通过 "Ti4+-IMAC富集磷酸肽、稳定同位素（二甲基）标记、LC-MS/MS检测"这一新磷酸化蛋白质组学定量分析技术路线，对人肝癌组织标本进行分析，检测出多个磷酸化蛋白（p-PED/PEA-15、p-P27-T187等）其磷酸化水平和程度可能参与调控癌细胞凋亡、转化和侵袭。在此基础上，本研究利用此磷酸化蛋白质组学定量分析技术检测人不同转移潜能肝癌细胞系磷酸化蛋白表达谱，高通量筛选与肝癌转移相关的磷酸化蛋白、磷酸化位点和激酶等信息；进一步挑选有意义差异磷酸化蛋白进行下游功能分析，利用点突变和特异性蛋白激酶抑制剂方法，观察去磷酸化修饰后其蛋白表达、细胞内分布以及对肝癌细胞转移特性的影响。旨在揭示肝癌转移复发的分子机制，寻找更好的肝癌预测转移指标和干预治疗靶分子。

本项目充分利用蛋白质组学定量分析技术平台，鉴定出迄今为止报道的最大规模人类肝脏组织磷酸化位点的数据集（共鉴定到了22446个磷酸化位点和6526个磷酸化蛋白质），并筛选了大量肝癌转移、耐药相关（磷酸化）蛋白质，探讨了其在肝癌转移、耐药中的作用及机制，为解决肝癌转移、耐药等瓶颈问题提供了重要理论依据。. 我们首先发展了酶辅助反相-反相液相色谱多维分离法等蛋白质组学技术，优化了肝癌蛋白质组学筛选平台，筛选出一系列肝癌转相、耐药关蛋白（bFGF、Hippo信号通路，Pinin、NgBR蛋白等）。在此基础上进行深入研究，探索并发现：二甲双胍可能通过Akt/GSK3β/Twist通路抑制bFGF诱导的肝癌侵袭与转移；二甲双胍可以通过AMPK/YAP信号途径促进肝癌化疗敏感性（Oncotarget. 2016, Jul 19）；NgBR能够影响p53泛素化，通过调控细胞进展影响肝癌细胞化疗药物耐药性（Oncotarget. 2016, Feb 23）；Pinin蛋白可以调节ERK磷酸化水平参与肝癌细胞糖耐受水平，在肝癌早期诊断、预后判断中具有重要意义（Oncotarget. 2016, Jun 28）。我们的工作不仅为进一步寻找肝癌预测转移、耐药的潜在生物标记物和干预治疗的靶分子提供了理论基础，同时也对二甲双胍在肝癌临床的治疗应用具有重要的理论指导和临床应用意义。