NRBP2对肝癌干细胞分化的调控及分子机制研究

Cancer stem cells (CSCs) have contributed to the tumorigenesis and development of cancer, and which are closely related to the cancer recurrence and metastasis. Differentiation therapy could inhibit the malignance of CSCs by the means of differentiation induction. In our previous study, we had demonstrated that exogenous BMP4 administration could induce CD133+ hepatic CSCs differentiation, and in our recent work, through the cDNA microarray analysis, we found that NRBP2 gene expression showed significant difference after the induction. Realtime PCR results showed that NRBP2 expression was up-regulated in the MHCC-97L cells after BMP4 treatment, and which was time-course dependent. NRBP2 expression in the CD133+ hepatic carcinoma (HCC) cells was down-regulated compared with its corresponding CD133- cells in the PLC/PRF/5, Huh7 and MHCC-97L cell lines. And knockdown of NRBP2 expression in MHCC-97L cells resulted in the expression up-regulation of the stemness related genes including Oct4, Tert and Ep300. All the results from our lab indicate that NRBP2 expression is closely related to the differentiation of CD133+ hepatic CSCs, though the function and regulation mechanism of NRBP2 affecting CSCs differentiation remain unclear. In this project, we plan to evaluate the function of NRBP2 in the hepatic CSCs differentiation using more cell lines, especially in vivo experiments. And with bioinformatics analysis, we plan to find out the target gene using cDNA microarray and protein array, and to explore the mechanism of NRBP2 affecting CD133+ hepatic CSCs differentiation. Then we will analysis the relationship of NRBP2 protein expression and the clinicopathologic features of HCC patients. Finally, we will expound the function and mechanism of NRBP2 in hepatic CSCs differentiation and provide molecular marker for the prognosis of liver cancer.

肿瘤干细胞对肿瘤的发生发展具有重要的促进作用，并且与肿瘤的复发和转移密切相关。分化治疗策略是通过诱导肿瘤干细胞分化，从而抑制肿瘤干细胞的恶性。我们先前的研究发现BMP4可以诱导CD133+肝癌干细胞分化。随后我们通过基因芯片分析，发现这一过程中NRBP2的表达具有显著改变。BMP4处理后肝癌细胞中NRBP2的表达增高，且呈时间依赖性；NRBP2在CD133-肝癌细胞中的表达高于CD133+细胞；干扰NRBP2基因表达会导致肝癌细胞中"干性"相关基因的表达增高。上述结果提示NRBP2与肝癌干细胞分化密切相关，但是其影响肝癌干细胞分化的分子机制并不明确。本研究拟结合肝癌患者临床标本，探索NRBP2与肝癌组织分化程度之间的相关性，并进一步明确NRBP2在肝癌干细胞分化中的功能，探讨NRBP2影响肝癌细胞分化的分子机制，为阐明肝癌干细胞分化机理提供实验依据，同时也为肝癌的预后判断提供分子标志。

系统性化疗是晚期肝细胞癌（Hepatocellular carcinoma，HCC）患者治疗的主要治疗选项，而耐药是导致HCC病人死亡的主要原因。最近的研究显示，肿瘤干细胞（Cancer stem cell, CSC）参与肿瘤的起始并在耐药中发挥重要作用。我们之前发现，利用BMP4诱导HCC中CD133+ CSCs发生分化，可以抑制CSCs的成瘤和耐药能力。在本项目中，通过分析诱导分化前后的HCC细胞中基因表达变化，我们发现核受体结合蛋白2（Nuclear receptor binding protein 2, NRBP2）基因在BMP4处理后表达增高。229例被检测的HCC组织和对应癌旁正常组织中，NRBP2在159例（69.43%）HCC组织中表达下调，67例（29.26%）表达无明显变化，仅在3例（1.31%）中表达增高。NRBP2表达与HCC患者肿瘤组织学分级呈负相关，NRBP2表达越低的患者预后越差。过表达NRBP2的HCC细胞中干性相关基因如Nanog、Oct3/4等表达下调，干扰NRBP2表达后这些基因则表达上调，NRBP2低表达细胞中富集了CSC细胞群。NRBP2高表达会抑制HCC的体外增殖、自我更新和体内成瘤能力，同时增强目前的一线化疗药物Sorafenib在HCC细胞中的生长抑制和促凋亡作用。生物信息学分析及体外实验的结果则显示，NRBP2高表达增强了HCC细胞对于Cisplatin和Akt抑制剂Perifosine的化疗敏感性。机制方面，NRBP2可以与Annexin A2（ANXA2）蛋白结合，并抑制ANXA2蛋白的表达。在过表达NRBP2基因的HCC细胞中同时过表达ANXA2，可以使得NRBP2过表达细胞的化疗药物敏感性、自我更新能力以及Akt磷酸化水平得以恢复。同时，我们发现NRBP2对HCC细胞化疗药物耐受能力的调控与其对Akt和Bcl2信号通路相关蛋白的表达有关。Perifosine处理可以抑制HCC细胞中干性相关基因表达和CD133+ CSCs在体外的自我更新，提示Perifosine可能也是HCC中CSCs的抑制剂。综上所述，我们的研究结果显示，结合在HCC组织中检测NRBP2的表达水平与Perifosine的选择使用，可能成为克服HCC病人化疗耐药的一种具有较好应用前景的治疗策略。