早老症患者EGR1表达升高的分子机制及其抵抗肿瘤转化功能研究

Genomic instability and subsequent accumulation of DNA damage are important risk factors for tumor inducing. However, the lack of tumors in progeroid patients may suggests some possible mechanisms of tumor transformation resistance. We studied a Han family with progeria caused by a LMNA gene mutation, and found that the EGR1 expression was up-regulated in progeroid patients. Recent study showed that the Arf-Egr-C/EBPβ axis was an important determinant of cellular responses to senescence or tumor transformation. Based on this, we proposed the hypothesis that the LMNA gene mutation can cause the epigenetic change which increase the EGR1 gene expression, and the elevated EGR1 expression level may play an important role in the tumor transformation resistance in patients with progeria. To prove this, firstly, we design experiments to investigate the epigenetics changes of EGR1 gene related to the LMNA mutation; and then, cultured fibroblast cells from the progeria patients or model mice will be transformed to immortalized tumor cells (TRS-progeria), and the transformed cells will be tested the tumorigenicity and EGR1 expression level. Finally, after knocking out (KO) of the EGR1 gene by CRISPR/Cas 9 in the progeria fibroblast cells, the EGR1 KO cells will be transformed into immortalized tumor cells (TRS-progeria-EGR1 KO) , and the clone forming ability and tumorigenicity of TRS-progeria-EGR1 KO cells will be tested and compared with TRS-progeria cells. This study will elucidate the molecular mechanism of EGR1 up-regulation and its function in the tumor transformation resistance, and will benefit to understand the tumor-protective mechanism of progeria.

儿童早老症患者基因组不稳定性增加却并不导致肿瘤发生，提示可能存在抵抗肿瘤转化的机制。我们发现一个LMNA基因突变导致的汉族儿童早老症家系，患者 EGR1表达显著升高。最近研究认为Arf-Egr-C/EBPβ调控轴决定细胞的衰老或者肿瘤转化。据此提出假说：LMNA基因突变可能导致表观遗传修饰改变而促进EGR1表达，而高表达EGR1可能通过促使细胞衰老而对肿瘤转化具有抵抗作用。为验证该假说设计研究：首先利用表观遗传学实验技术检测LMNA突变是否导致EGR1基因所在的染色质区域定位改变及其组蛋白修饰改变；然后体外培养获得早老症模型成纤维细胞并将其进行肿瘤转化，检测转化后细胞的致瘤性和EGR1表达；最后通过CRISPR/cas9技术敲除早老症患者成纤维细胞内EGR1基因，再将敲除细胞系进行肿瘤转化，比较其克隆形成能力和致瘤性改变情况。本研究将阐明EGR1表达上升的机制及其抵抗肿瘤转化的功能。

儿童早老症与生理性衰老有较多相似之处，包括细胞核变形、组蛋白修饰改变和基因组不稳定性增加等，对儿童早老症的致病机制研究有助于人们理解生理性衰老。我们在发现广西第一例LMNA R527C突变型儿童早老症家系后，接连又发现了两个该突变类型儿童早老症家系。由于突变类型一样，又同属于广西人群（一个家系为瑶族，另外两个家系为汉族），该疾病临床表现几乎一致。三个家系基因组STR（short tandem repeats）分析显示该突变来自共同祖先。我们利用免疫组织化学方法分别检测了三个家系患者皮肤样本，都不同程度的发现皮肤真皮组织EGR1表达升高，三个家系6名患者随访观察也均未发现肿瘤发生。我们通过转化外周血白细胞获得了患者的诱导多能干细胞（iPS），并体外诱导分化获得了间充质干细胞（iPS-MSC），发现患者来源的iPS-MSC较正常来源iPS-MSC生长缓慢，P16表达明显升高，具有明显的抵抗肿瘤转化特性。为了研究LMNA基因功能，我们利用CRISPR/cas9技术敲除了HepG2细胞和293T细胞内LMNA基因，发现这两株LMNA完全敲除细胞内P16表达上调，克隆形成能力和裸鼠体内致瘤性都下降。在LMNA KO细胞内表达致病突变体LMNA R527C基因，发现LMNA R527C突变基因影响细胞核刚度。儿童早老症患者抵抗肿瘤的特性不仅与细胞特性相关，可能也与机体免疫系统的异常或者过度激活相关，根据这个推测，我们利用单细胞测序技术分析了5名患者的外周血细胞，发现所有患者巨噬细胞异常，这是否也是儿童早老症患者抵抗肿瘤的原因有待进一步研究。