p65调控WDR79转录促进肺癌发展和耐药作用及机制

Studies revealed that the NF-κB signaling pathway was in continuous activated state in a series of tumors, and inhibition of the NF-κB signaling pathway can block the cell cycles and induce apoptosis. p65 protein is a subunit of NF-κB. It was found that p65 could precisely control the transcription activatity of NF-κB and played an important role in occurrence, development and drug resistance of cancer. Therefore, targeting the transcription of downstream genes regulated by p65 is becoming a new strategy for developing anti-cancer drugs. WDR79 belongs to the family of WD repeat proteins and is over-expressed in various cancer cell lines. But little is known about the biological function and mechanism of WDR79 in tumor. Recently, we found that knock-down WDR79 effectively surpressed the cell proliferation and promoted the cell apoptosis. Moreover, our preliminary data suggested that the change of WDR79 protein expression level was not affected the level of p65 protein. Furthermore, WDR79 expression was reduced significantly when depletion of p65. We have analysed the WDR79 promoter area on jaspar website. The results showed that there were two potential binding sites of p65 protein in WDR79 promoter. Together, we assumed that p65 promoted cancer progression and drug resistance by regulating the transcription of WDR79 through binding to WDR79 promoter. Verify of this science hypothesis will help to elucidate the molecular mechanism of WDR79 promoting cancer progression and drug resistance and will provide new clues for the research of anti-cancer drug.

大多数肿瘤细胞中NF-κB信号通路处于持续激活状态，抑制该信号通路能够阻断细胞周期，诱导凋亡。p65是NF-κB的一个亚单位，能够精细地调控NF-κB的转录激活，在癌症的发生、发展和耐药中发挥重要的作用。因此，研究p65蛋白对下游基因的转录调控成为开发靶向p65蛋白抗肿瘤药物的新策略。WDR79隶属于WD重复蛋白家族，在多种肿瘤中高表达，但是其在癌症发展和耐药中的生物学功能和作用机制尚知之甚少。我们发现敲低WDR79表达能够有效抑制肿瘤生长，而p65蛋白表达水平未发生变化。敲低p65表达，能够显著降低WDR79的表达量。利用jaspar网站分析WDR79启动子区域，发现有2个潜在的p65蛋白的结合位置。这些促使我们推测p65通过与WDR79启动子结合调节其转录，从而促进癌症发展和耐药。本项目对以上假说的验证有助于阐明WDR79促进癌症发展和耐药的机制，为抗癌药物研发提供新的思路。

WDR79是WD重复蛋白家族一员，在进化中高度保守，是细胞核内卡哈尔体形成必不可少的成分。之前的研究发现WDR79能够促进非小细胞肺癌细胞增殖和恶性转化，为了找寻WDR79上游基因，通过软件分析出p65蛋白是WDR79蛋白潜在的上游调控基因。通过蛋白免疫印迹实验，发现 p65和WDR79蛋白在肺癌细胞中的表达呈现相关性，但是免疫组化实验显示肺癌临床样本中p65和WDR79蛋白表达无明显相关性。开展了转录因子 WDR79 的生物信息学分析，从基因启动子序列入手分析了 WDR79的结构和潜在功能预测，同时分析 WDR79潜在的上下游基因。通过RNA干扰实验，证实在A549细胞中p65是WDR79上游调控基因，并通过染色质免疫共沉淀实验发现p65蛋白WDR79启动子存在物理性作用。同时构建了WDR79启动子序列质粒，并通过荧光素酶实验验证了所构建质粒的效果。我们的结果揭示了p65能够调控WDR79基因促进了癌症的发展，为防治非小细胞肺癌提供了新的治疗策略。在本项目的资助下，我们已经发表论文6篇，培养硕士研究生3名（已毕业），11名本科生（已毕业）。