DNA羟甲基化在LASS2介导的胶质瘤增殖和侵袭中的作用及机制研究

The important factor for the failure of clinical therapy is that glioma has the invasive behaviour. Longevity assurance homolog 2 of yeast LAG1 (LASS2) is a cancer suppressor gene. Our previous study showed that LASS2 was significantly down-regulated in malignant gliomas and its level was closely related with patients’ prognosis. In addition, comparing with control cells, the proliferation and invasion of U87MG cells with over-expression of LASS2 were significantly decreased, suggesting LASS2 may be a cancer suppressor gene in gliomas. However, the precise mechanism of LASS2 downregulation remains unclarified. Recent study reveals that there are LASS2 gene promoter methylation in glioma specimens, and there are positive relationships between LASS2 and Tet1 and 5hmC in protein level. Besides, when Tet1 is downregulated by targeting interference can decrease LASS2 expression. These results reveal that there are close relationship between LASS2 and Tet1. Therefore, we attempt to investigate: 1) the role of LASS2 gene methylation/ hydroxymethylation regulation in the proliferation and invasion of glioma, and 2) the mechanism of Tet1 regulation in LASS2 gene hydroxymethylation. It is aimed to insight the mechanism of LASS2 regulation of the proliferation and invasion in glioma and offer new potential therapeutic implications.

增殖失控和弥漫浸润是阻碍胶质瘤临床治疗的关键因素。Longevity assurance homolog 2 of yeast LAG1 (LASS2) 基因被认为是一种抑癌基因。我们前期研究发现LASS2在胶质瘤中表达明显下降，与病人预后密切相关；且LASS2过表达后，U87MG细胞增殖和侵袭能力明显降低，提示LASS2对胶质瘤具有抑癌作用，但LASS2表达下调的调控机制不清。前期结果显示：1）在胶质瘤标本中LASS2基因存在甲基化；2）免疫组化示LASS2与羟甲基化酶Tet1以及5hmC的表达呈正相关；3）干扰Tet1后，LASS2表达降低，提示LASS2与羟甲基化密切相关。因此，本课题拟研究：LASS2甲基化/羟甲基化调控在胶质瘤增殖和侵袭方面的作用和机制；Tet1调控LASS2基因羟甲基化的内在机理。希望能深入探讨LASS2介导胶质瘤增殖和侵袭的分子机制，为治疗胶质瘤提供新思路。

增殖失控和弥漫浸润是阻碍胶质瘤临床治疗的关键因素。LASS2基因被认为是一种抑癌基因。结果： 第一部分，发现1）与正常脑组织相比，LASS2在胶质瘤中表达明显下降，与病人预后密切相关（P<0.05）；2）LASS2过表达后，U87MG细胞增殖和侵袭能力明显降低，提示LASS2对胶质瘤具有抑癌作用；3）在胶质瘤标本中LASS2基因存在甲基化；免疫组化示LASS2与羟甲基化酶Tet1以及5hmC的表达呈正相关；4）细胞株中，干扰Tet1后，LASS2表达降低；过表达Tet1后，LASS2表达增加，提示TET1可调控LASS2表达，减少CyclinD1的表达，抑制肿瘤细胞增殖；5）ChIP和HMeDIP分析显示TET1可羟甲基化调控LASS2基因启动子区5-hmC的表达。.第二部分，在胶质瘤中常发生IDH突变，IDH突变导致TETs催化胞嘧啶去甲基化能力下降，5hmC生成减少。6）弥散张量MRI结果显示发生IDH突变的胶质瘤具有较高的最小ADC值和较低的最大FA值；7）IDH1/2突变的胶质瘤标本，其肿瘤细胞的增值指数和血管微密度都要明显低于IDH野生型的标本（P<0.05），提示IDH突变可抑制肿瘤细胞的增值和血管生成。8）发生1p/19q杂合性缺失或者IDH1/2突变的病例多位于额叶（P<0.05）和岛叶，并且在T1WI强化像上多没有强化或仅轻度强化；9）联合标化最小ADC值和常规MRI评估少突胶质瘤病例有无IDH突变可获得92.2%的敏感性、75.8%的特异性、93.8%的阳性预测值和71.1%的阴性预测值。结论： LASS2高表达的胶质瘤病人预后较好；TET1羟甲基化调控LASS2基因启动子区5-hmC的表达，导致CyclinD1的表达下降，抑制肿瘤细胞增殖；弥散张量MRI参数可以提供一种无创性评估胶质瘤的IDH突变情况，为诊断和治疗胶质瘤提供新思路。