核蛋白FUBP2对肺癌转移的影响及分子机制研究

Metastasis is the main cause of death in patients with lung cancer. However, the precise molecular mechanisms have not been sufficiently analyzed. Our previous study identified metastasis-associated proteins that were differentially expressed between lung adenocarcinoma cancer cell lines SPC-A-1sci cells with high metastatic potential and parent SPC-A-1 cells with low metastatic potential. Here, metastasis-associated nucleoproteins of these two cells were analyzed by using the isobaric tags for relative and absolute quantitation and SWATH approach combined with nano liquid chromatography-tandem mass spectrometry analysis. The protein, far upstream element-binding protein 2 (FUBP2), was validated to be a potential novel biomarker and was chosen to be a candidate metastasis-associated nucleoprotein. we focus on the effects of FUBP2 on metastasis of lung carcinoma by in vitro and in vivo, identify the protein molecules which was interacted with FUBP2 by using co-immunoprecipitation and proteomic analysis, determine their interaction domain or motif, verify functions and effects of the interaction protein in promotion of metastasis, elucidate the molecular mechanism of metastasis, validate its clinical significance in a mass of lung cancer specimens. This study will help to elucidate the molecular mechanism of metastasis, to find new targets for intervention and to provide a novel idea for diagnosis and treatment of lung carcinoma.

转移是肺癌患者死亡的主要原因,但肺癌转移机制尚未完全阐明。本实验室前期通过体内筛选建立了拥有自主知识产权的肺癌高、低转移细胞株，并分析了二者之间总蛋白及分泌蛋白的表达差异。该项目进一步以核蛋白为切入点，采用两对肺癌高低转移细胞，两种蛋白质组学方法，结合小样本临床肺癌组织验证，筛选鉴定出远端上游元件结合蛋白2（FUBP2）作为肺癌转移相关候选蛋白；通过体外、体内实验研究FUBP2在肺癌转移中的作用；通过免疫共沉淀、蛋白组学分析及基因表达谱芯片分析确定其相互作用蛋白，确定相互作用结构域/基序；明确该蛋白的功能及在FUBP2促肺癌转移中的作用；明确FUBP2与相互作用蛋白对C-myc基因的调节作用及其作用的下游信号传导通路，阐明FUBP2影响肺癌转移的分子机制；研究FUBP2及相互作用蛋白的临床意义。该项目的顺利完成将为阐明肺癌转移的机理，寻找新的干预靶点及发展新的治疗策略提供理论与应用依据。

转移是肺癌患者死亡的主要原因,但肺癌转移机制尚未完全阐明。本实验室前期通过体内筛选建立了拥有自主知识产权的肺癌高、低转移细胞株，并分析了二者之间总蛋白及分泌蛋白的表达差异。该项目进一步以核蛋白为切入点，采用两对肺癌高低转移细胞、两种蛋白质组学方法，结合小样本临床肺癌组织验证，筛选鉴定出远端上游元件结合蛋白2（FUBP2）(又名KH型剪切调节蛋白，KHSRP)作为肺癌转移相关候选蛋白。在体外及体内实验研究中，证实FUBP2的基因沉默对肺癌细胞的增殖、迁移和侵袭有明显的抑制作用，而FUBP2的过表达对肺癌细胞的增殖、迁移和侵袭有明显的促进作用。通过免疫共沉淀实验结合蛋白质谱分析，鉴定出HNRNPC蛋白可能与FUBP2相互作用，分别在内源性及外源性表达FUBP2和HNRNPC的细胞中，通过免疫共沉淀及Western blot实验证实HNRNPC能够与FUBP2相互作用。在体外研究中，证实HNRNPC的基因沉默抑制了肺癌细胞的体外增殖、迁移和侵袭，而HNRNPC的过表达则促进了肺癌细胞的体外和体内增殖、迁移和侵袭。FUBP2和HNRNPC通过作用JAK-Stat信号传导通路从而调控肺癌转移。FUBP2和HNRNPC在肺癌组织中的表达明显高于癌旁组织，FUBP2和HNRNPC表达增高与肿瘤分期、淋巴结转移及远处转移密切相关。Kaplan-Meier生存分析显示，高FUBP2和HNRNPC表达的肺癌患者预后较差。我们的发现提示FUBP2可以作为一种新的预后生物标记物，并为临床实践和个性化治疗决策提供一个潜在的治疗靶点。