胸腺肽β4介导循环肝癌细胞抵抗失巢凋亡的分子机制研究

The magnetic bead separation coupled with MALDI-TOF MS is increasingly being applied to the areas of biomarker discovery. Using this novel system, we have found a distinguished differentially expressed protein peak in sera of patients with hepatocellular carcinoma (HCC), which was identified as thymosin β4 (Tβ4). It has been known that Tβ4 is a major actin-binding protein, rather than a true thymic hormone, which is suggested to be closely related to tumor development and metastasis. However, until now, there isn’t any data regarding the relationship between Tβ4 and HCC. We performed a variety of experiments with the different samples from HCC cell lines and tissues, these studies indicated that Tβ4 as a potential diagnostic and prognostic marker in patients with HCC. Tβ4 not only could promote cell proliferation, migration, and invasion in HCC, but also upregulate the tyrosine kinase receptor B（TrkB）and display more resistance to anoikis. Unfortunately, little is known about the survival mechanism of CTC in HCC. Based on our results and the existing literature, we propose a hypothesis for the regulation mechanisms of CTC survival that hepatoma cells with high expression Tβ4, on the one hand, indirectly accumulation of β-catenin in the cytoplasmic then incorporation it to nuclear, which can activate nuclear transcription factors and promote cell proliferation and survival. On the other hand, Tβ4, a cytokine present in HCC cells and tumor microenvironment, activate the PI3K/Akt pathway via integrin linked kinase (ILK), and subsequently activate the nuclear factor κB (NF-κB) survival pathway, mediating apoptotic proteins in cancer cells, which results in the acquisition of more survival, and have an increased ability to migrate into the bloodstream and display more resistance to anoikis. Based on our novel detection system for HCC CTC (asialoglycoprotein receptor-based magnetic cell separation combined with anti-P-CK and anti-carbamoyl phosphate synthetase 1 (CPS1) antibodies staining detection), In addition to collection of further experimental evidence for Tβ4 induced anoikis resistance, this project will focus on hypothesis verification by using signalling pathway specific inhibitors, neutralizing antibodies, and interfering RNA to shut down a signaling pathway, finally figuring out how Tβ4 mediates the anoikis resistance by upregulation of TrkB in HCC.

胸腺肽β4（Tβ4）是一个肌动蛋白隐蔽蛋白，与肿瘤关系密切。我们的研究结果表明，Tβ4是一个潜在的肝癌标志物，并且能显著诱导肝癌细胞失巢凋亡抵抗。循环肿瘤细胞（CTC）是肝癌复发转移的一个主要根源，而获得失巢凋亡抵抗是CTC扩散和转移的一个先决条件。因此，阐明肝癌CTC获得失巢凋亡抵抗并存活的机制具有重要的临床意义。根据前期试验和现有知识，我们推测肝癌CTC高表达Tβ4不仅间接使胞质内的β-catenin积聚并入核，而且通过ILK激活PI3K/Akt/NF-κB通路，NF-κB也可上调TrkB转导PI3K/Akt信号，从而形成一个正反馈调节环。本申请书设计了针对性研究内容，通过体外细胞试验和体内动物试验的研究，重点验证上述推测。期望能够阐明Tβ4诱导肝癌CTC存活和失巢凋亡抵抗的交互信号通路和分子机制，为发展一个基于靶向CTC失巢凋亡的肝癌复发转移治疗新模式提供理论基础和实验依据。

胸腺肽β4（Tβ4）是一个肌动蛋白隐蔽蛋白，与肿瘤关系密切。我们的研究结果表明，Tβ4是一个潜在的肝癌标志物，并且能显著诱导肝癌细胞失巢凋亡抵抗。循环肿瘤细胞（CTC）是肝癌复发转移的一个主要根源，而获得失巢凋亡抵抗是CTC扩散和转移的一个先决条件。因此，阐明肝癌CTC获得失巢凋亡抵抗并存活的机制具有重要的临床意义。. 本研究构建带GFP荧光标签的Tβ4表达慢病毒载体，分别感染Huh7、SMMMC-7721两个肝癌细胞系、筛选出稳定高表达Tβ4的肝癌细胞。以筛选得到的稳转细胞为实验对象，通过体外实验研究循环肝癌细胞抵抗失巢凋亡的分子机制。得到如下重要结果：（1）Tβ4 可以显著促进肝癌细胞增殖、迁移、侵袭能力。（2）Tβ4 能赋予肝癌细胞抵抗失巢凋亡的能力。（3）Tβ4抵抗失巢凋亡主要是由于Tβ4直接上调、激活ILK，从而激活PI3K/Akt/NF-κB通路，从而使肝癌细胞具有失巢凋亡抵抗能力得以存活。另外肝癌细胞高表达Tβ4，激活PI3K-Akt，使GSK3β失活，而GSK3β失活不能将细胞质内积聚的β-catenin磷酸化，使β-catenin降解受阻，从而使胞质内的β-catenin积聚并入核，激活核内转录因子，从而促进细胞增殖和存活。（4）通过特异性抑制剂MK2206抑制PI3K/Akt，能使细胞的失巢凋亡率增加，失巢凋亡抵抗标志物TrkB表达减少，凋亡蛋白caspase3表达上调，抗凋亡蛋白Bcl-2表达降低。另外Akt的磷酸化被抑制后，p-GSK3β表达升高，而β-catentin和NF-κB的表达减少。（5）临床资料样本统计得出：Tβ4的表达与年龄、性别、肿瘤分化程度、肿瘤大小及HBsAg无显著相关性，然而Tβ4的表达与临床分期和血管侵犯存在显著相关性，而且高Tβ4表达的HCC患者有更高的晚期和血管侵犯倾向；另外生存分析结果显示Tβ4阳性肝癌患者的总生存率明显低于Tβ4阴性肝癌患者，无复发生存率分析也显示了类似的结果。这些研究结果初步阐明Tβ4诱导肝癌CTC存活和失巢凋亡抵抗的分子机制，为Tβ4上调作为肝癌的一个恶性标志物提供理论基础；同时Tβ4可作为一个潜在的肝癌标志物对于肝癌具有诊断和预测预后的临床价值。. 增加的相关研究内容：肝癌患者循环肿瘤细胞PD-L1表型鉴定及临床意义的研究。