负性协同刺激分子B7-H4的核转移促进肿瘤发展的作用机制研究

It is known that as the immune checkpoint the negative costimulatory molecule B7-H4 is highly expressed in tumor and plays an important role in tumor immune evasion through inhibiting T cell proliferation.However previously we found that B7-H4 could express in nucleus in renal cell carcinoma and was correlated with tumor stage. Further investigation found that human B7-H4 contained not only signal peptide but also nuclear localization sequence (NLS). Subsequent investigation identified that B7-H4 could promote tumor cells proliferation through upregulating the expression of Cyclin D and Cyclin E.It has been found that many dual localization proteins can bind to promoters and regulate gene expression through the formation of a complex with other transcription factors.We predicted that B7-H4 might act through a similar mechanism. Based on this hypothesis, we plan to identify the target genes that are regulated by nuclear B7-H4 using approaches such as proteomics and Chromatin Immunoprecipitation-Sequence(ChIP-Seq). Furthermore we will investigate which transcription factors are involved in the formation of the complex with B7-H4 by immunoprecipitation. Through the implementation of this project we would like to elucidate the exact mechanism by which B7-H4 promotes tumor cell proliferation and propose a new theory about the effect of costimulatory molecule in tumor progression. This work will also pave a new avenue for the therapeutic treatment of renal cell carcinoma.

已知负性协同刺激分子B7-H4作为免疫调节的卡控点异常高表达于肿瘤,参与肿瘤的免疫逃逸,但确切机制尚待深入探讨。课题组前期研究临床肾癌标本发现B7-H4可定位于细胞核，并与临床分期相关，进而发现人B7-H4分子不仅含信号肽序列，还有核定位序列，核转移B7-H4可上调Cyclin D的表达促进肿瘤细胞增殖(Oncogene 2013)。基于许多双定位蛋白在细胞核中都能与其他转录因子结合形成转录复合物调控基因表达,课题组推测B7-H4可能具有类似作用机制，为证实这一科学假说，课题组计划以肾癌为研究模型，结合临床样本、转基因细胞株，通过体外、临床及动物实验，运用蛋白组学法和染色质免疫共沉淀-测序法，系统研究B7-H4在细胞核内调控表达的目的基因及结合的转录因子，阐明B7-H4核转移促进肿瘤发展的确切分子机制，为协同刺激分子功能的研究及设计特异性的肿瘤治疗新途径提供新的理论基础。

B7-H4分子包含核定位序列，可转移到细胞核。但是其核转移的生物学意义目前还不知。在本项目的研究中，我们通过免疫共沉淀及LC-MS/MS技术找到与B7-H4相互作用的蛋白，并鉴定出DNA-PKcs是其中一个互作蛋白。B7-H4可抑制zeocin诱导的DNA-PKcs的磷酸化，并发现B7-H4可抑制DNA损伤修复中的NHEJ通路。但是，通过克隆生存率及γH2AX foci实验显示B7-H4可促进DNA损伤的双链修复（DSB）。由于DSB修复主要包括HR和NHEJ两条通路，我们分析RAD51和BRCA1聚焦，实验结果显示B7-H4可促进DSB修复，并通过促进HR通路，抑制NHEJ通路来达到修复选择，这也可能是今后肿瘤治疗策略制定中考虑的一个因素。