EB病毒LMP1活化NF-kB下调PinX1激活端粒酶在EBV致鼻咽癌进程中的分子机制

Aberrant enhancement of telomerase activity by Epstein-Barr virus infection is believed to be a critical step in the early stage of carcinogenesis in nasopharyngeal carcinoma. Human PinX1, a potent tumor suppressor gene, has been elucidated as a novel telomerase inhibitor. However, the mechanism how EBV controls the activity of telomerase through PinX1 gene remains unclear. Our preliminary result showed that down-regulation of PinX1 expression in nasopharyngeal carcinoma was associated with aberrant CpG island hypermethylation of PinX1 gene. Furthermore, EBV infection could facilitate the degree of CpG island hypermethylation of PinX1. Moreover, DNMT3b has been identified as a novel target gene of NF-κB by our previously functional research. Finally, we clarify that LMP1, the oncoprotein of EBV, could upregulate DNMT3b expression and subsequently inhibit PinX1 expression through activation of NF-κB. On the other hand, two NF-κB binding sites in the 5’ flaking region of PinX1 gene have been predicted by bioinformatics analyses. On the basis of our preliminary results, we will demonstrate that PinX1 will be a novel target gene of NF-κB. Moreover, “LMP1/NF-κB/DNMT3b/PinX1” and “LMP1/NF-κB/PinX1”, which are two novel signaling pathways, could promote telomerase activity. Our further study will confirm that the role of these two novel signaling pathways in EBV LMP1-mediated enhancement of telomerase activity will be clarified in the process from oncogenic transformation of nasopharyngeal epithelia to the formation and development of nasopharyngeal carcinoma. Our research will provide new targets for prediction and therapy in Nasopharyngeal carcinoma, especially in nasopharyngeal carcinoma high-risk population who have been persistently infected with EBV, which will provide the profound scientific implication and superb value for clinical application.

EBV激活端粒酶是EBV致鼻咽癌早期关键步骤，PinX1是抑制端粒酶活性的抑癌基因，目前EBV通过PinX1调控端粒酶机制未明。我们已证实：EBV感染促进PinX1甲基化程度，PinX1异常高甲基化致其表达下调，DNMT3b是NF-κB调控的新靶基因，过表达LMP1可激活NF-κB上调DNMT3b以甲基化调控抑制PinX1表达；进一步预测PinX1启动子区也存在NF-κB结合位点。基于此提出本项目科学假说：PinX1可能是NF-κB调控的新靶基因，LMP1/NF-κB/DNMT3b/PinX1和LMP1/NF-κB/PinX1可能是活化端粒酶促进细胞增殖的两条新通路。拟构建EBV感染细胞动态转化模型，明确两通路在EBV致鼻咽癌不同阶段激活端粒酶对细胞功能的影响；以通路中关键分子为靶点诱导EBV进入裂解期并清除EBV感染细胞，为EBV感染致癌高危人群和鼻咽癌患者实施预测和有效治疗探索新思路。

EBV激活端粒酶是EBV致鼻咽癌早期关键步骤，PINX1是抑制端粒酶活性的抑癌基因，目前EBV是否通过PINX1调控端粒酶促进鼻咽癌发病的机制未明。因此本项目围绕PINX1在鼻咽癌中的表达、功能和分子机制展开研究。.明确端粒酶抑制因子PINX1在鼻咽癌中的表达水平和生物学功能，从突变、表观遗传学修饰（DNA甲基化）以及转录调控多方面探讨PINX1在鼻咽癌中异常表达的分子机制。首先通过免疫组织化学实验和RT-PCR、western blot方法，从组织和细胞水平明确端粒酶抑制因子PINX1在鼻咽癌中相对低表达。其次，从DNA甲基化和基因突变两方面深入挖掘PINX1在鼻咽癌中低表达的调控机制。通过BSP，MS-HRM，去甲基化的药物处理等方法在鼻咽癌细胞中证明了甲基化调控可能并非致PINX1下调的主要原因。外显子测序分析，结果提示PINX1的良性多态性与鼻咽癌中PINX1的低表达无明显相关性。生物信息学预测PINX1的转录调节因子，发现其启动子区可能存在潜在的NF-κB和STAT3结合位点。我们进一步从转录调控方面探讨了PINX1表达异常的分子机制以及在鼻咽癌中生物学功能。.研究证实PINX1为NF-κB和STAT3新的靶基因。EB病毒LMP1可活化转录因子NF-kB、STAT3，从而负性转录调节 PINX1表达，激活端粒酶，促进鼻咽癌增殖、侵袭。同时在实验过程中发现PINX1具有负性调控NFκB活性表达的作用。因此，研究进一步延伸，在EBV诱导鼻咽癌发生发展过程中NFκB与PINX1形成反馈环路致NFκB持续活化，促进鼻咽癌转移和化疗抵抗的分子机制的构思和探索。拟将通过细胞体外实验及体内动物治疗实验证实PINX1负性调控NFκB能抑制鼻咽癌转移和化疗抵抗。如果得以证实预示着PINX1是打破这个恶性负反馈环路的关键靶点。.根据PINX1在鼻咽癌中的表达和功能机制，预示着PINX1可为鼻咽癌潜在的治疗靶点。目前，针对PINX1为靶点的临床药物未见文献报道。本项目将为后续PINX1靶向药物的创新性研究奠定基础和提供理论证据。