组蛋白H3K4/27甲基化异常参与NSCLC细胞“干性”获得及维持的机制研究

Histone methylation modification is abnormally involved in the occurrence, development and pathological outcom of cancer, while lung cancer stem cells are believed to be the source of lung cancer recurrence and metastasis. We have previously found that the level of 5-hmC is low in NSCLC compared with the corresponding adjacent normal tissues, moreover, loss of 5-hmC was revealed to be associated with the poor prognosis of NSCLC patients (Oncol Lett. 2016). Recently, with the immunohistochemistry analysis, we showed up-regulated level of H3K4me3 and down-regulated H3K27me3 were founded in NSCLC compared with the para-tumorous normal lung tissues, and the high level of H3K4me3 and low level of H3K27me3 were associated with the poor prognosis of NSCLC patients. And these abnormal H3K4/27 methylations were reported to participate in the acquisition and maintenance of the characteristic of stem cells. Based on this relationship, we speculated that abnormal H3K4/27 methylation promote the development of NSCLC by endowing and maintaining lung cancer cells with stemness characteristic. Here, we will try to reveal the relationship among the level of H3K4/27 methylation, lung cancer stem cells and the prognosis of NSCLC patients in NSCLC tissues. With regulating the level of H3K4/27 methylation, we will further determine the effect of H3K4/27 methylation on the biological function and the stemness acquisition and maintenance of lung cancer cells. The detailed genes and key pathway regulated by H3K4/27 methylation will be studied by RNA/mChIP-seq. Finally, the in vivo study will be employed to validate the above results. Our study here will reveal the abnormal H3K4/27 methylation in the acquisition and maintenance of lung cancer cell stemness, potentially providing a new therapeutic target for lung cancer.

组蛋白甲基化修饰异常参与肿瘤发生、发展和病理转归；而肺癌干细胞被认为是肺癌转移复发的根源。我们既往证实DNA甲基化修饰5-羟甲基化胞嘧啶在肺癌组织中呈低水平，且其低水平组织的患者预后差（Oncol Lett. 2016）。我们近期在肺癌细胞与组织中发现H3K4me3升高而H3K27me3降低，且有两者这一变化的患者预后最差。结合文献报道两者参与成体细胞干性获得与维持。故推测组蛋白H3K4/27甲基化异常通过参与肺癌细胞干性获得与维持促肺癌进展。课题拟在组织中明确H3K4/27me3、相关甲基化酶、肺癌干细胞标记及与患者预后的关系；细胞中阐明调节H3K4/27甲基化对肺癌细胞生物学功能、干性获得及维持的影响；分子水平用RNA/mChIP-seq等明确H3K4/27甲基化异常促肺癌细胞干性获得与维持的关键基因与信号；最后动物水平验证。课题将揭示表观修饰与肺癌干细胞间关系，为肺癌治疗提供新靶点。

表观遗传参与肿瘤的发生和发展，包括非小细胞肺癌（NSCLC和食管癌），使肿瘤适应各种微环境的变化。其中，组蛋白3赖氨酸4（H3K4）的甲基化修饰参与调控多种肿瘤的发生和发展，包括单甲基化（H3K4me1）、二甲基化（H3K4me2）和三甲基化（H3K4me3），因为其募集转录因子/共激活因子的能力，通常被认为是转录激活的标志。由于参与H3K4甲基化修饰的甲基化酶和去甲基化酶种类较多，可以相互作用，且表观遗传的阅读器（Readers）也参与下游基因的调控，故此系统调控NSCLC和食管癌进展的机制是十分复杂的。本课题在前期工作基础上，证实：（1）ING4在大部分NSCLC组织中表达低于癌旁组织，且与NSCLC患者临床病例特征和预后及复发相关。单因素和多因素分析发现ING4是影响NSCLC预后的独立危险因素，ING4可作为NSCLC预后预测的潜在的生物标志物。（2）H3K4me3在大部分NSCLC组织中表达高于癌旁组织，且与NSCLC患者预后及复发相关。（3）在NSCLC细胞中改变ING4表达水平，发现敲减ING4可以促进NSCLC细胞的增殖、迁移及侵袭能力，促进NSCLC干性的获得和维持，体内实验验证了敲减ING4可以促进NSCLC的进展。（4）ChIP-seq和RNA-seq发现ING4/ H3K4me3通过调控下游的癌基因TWIST1的表达来影响NSCLC的进展。（5）NSCLC患者中，ING4表达水平与H3K4me3水平呈负相关，且低表达ING4和高表达H3K4me3组患者的预后较高表达ING4和低表达H3K4me3组的患者更差。另外，本课题研究还揭示食管癌（HEC）患者中H3K4me3水平较癌旁组织明显升高，且高水平H3K4me3与HEC患者临床病例特征和预后及复发相关。在HEC细胞中敲除ING4后，HEC细胞的增殖、转移、侵袭和增殖增强。高水平的H3K4me3可作为HEC预后预测的潜在的生物标志物。