Tet1介导的全基因组5hmC分布谱异常在髓母细胞瘤发病机制中的作用研究

Medulloblastoma (MB), the most common brain malignancy in children, is classified into four subgroups: WNT, SHH, Group 3, and Group 4. Preliminary data shows：genomic 5-hydroxymethylcytosine is significantly depleted in all subgroups of MB. TET1 levels in MB are similar to the levels in fetus but much higher than adult levels, while there is no significant change in the expression of TET2 and TET3. SmoA1 mice are a spontaneous medulloblastoma mouse model recapitulating human SHH medulloblastomas. To test whether elevated TET1 is essential for MB survival, we treated a STAT/TET1 axis inhibitor (UC-514321) to SmoA1-derived primary MB cells. In contrast to neuronal stem cells (NSC), the inhibitor treatment led to a decrease in cell viability of primary MB cells. Our preliminary analyses suggest that the heterozygous KO of TET1 crossed with SmoA1 mice led to an increase in symptom free survival. Given these data, we conclude that Tet1 functions as an oncoprotein by creating a tumor-specific 5hmC pattern. Murine SHH MB could be treated by inhibiting TET1. In the next stage, we will focus on human MB tissues. 1) Subtypes of these MBs will be tested by IHC. Primary MB cells will be treated with UC-514321. Primary MB cells will be used for tumorigenesis test in immunodeficient mice. By testing the cell viability and the progression of MB tumor in mice, we will confirm which subtypes of human MB could be treated by inhibiting TET1; 2) By genomic 5hmC-seq, we will characterize unique genome-wide 5hmC patterns for each subgroup and perform meta-analysis with public transcriptome data; 3) By up- and down-regulating TET1 in human MB cells, RNA-seq will be performed to find the targeted genes of TET1 to further understand the mechanism of Tet1-mediated modulation.

髓母细胞瘤（MB）是最常见的儿童恶性脑肿瘤，分为WNT型、SHH型、3型和4型。我们发现MB全基因组5羟甲基化胞嘧啶（5hmC）水平下降，且TET1表达增加，提示TET1可能参与致病。应用MB特异性的TET1抑制剂:UC-514321处理SHH型MB模型SmoA1小鼠肿瘤细胞，其活性明显下降。TET1 KO小鼠与SmoA1小鼠杂交，后代25周无瘤生存率由25%升至75%。本研究将继续探索TET1介导的全基因组5hmC分布谱异常在不同亚型患者MB中的作用。鉴定并培养各亚型患者MB，用UC-5143321处理原代MB细胞及成瘤裸鼠，探讨其对不同亚型人类MB的治疗作用。对MB进行全基因组5hmC测序和RNA-seq，明确不同亚型MB特异性5hmC分布谱。调节MB的TET1水平，运用5hmC-seq和RNA-seq，寻找TET1靶基因，明确Tet1介导的5hmC分布谱异常在MB发病机制中的作用。

髓母细胞瘤是最常见的恶性儿童脑肿瘤，起源于小脑蚓部。频繁的髓母细胞瘤体细胞突变及表观遗传学调节因子的下调提示表观遗传学变化起到非常重的作用。5羟甲基胞嘧啶（5hmC）在小脑的发育过程中是很常见的胞嘧啶调节的中间产物，该过程受TET酶家族的调控。本项目研究了髓母细胞瘤中5hmC和TET酶家族的变化以及它们在小脑肿瘤形成过程中所起到的作用。我们的实验结果证实，与正常小脑相比，无论是人类的髓母细胞瘤，还是模型小鼠生成的髓母细胞瘤，全基因组DNA中的5hmC全部下调。但同时，全基因组测序发现髓母细胞瘤中，在某些具有类似于干细胞属性和Nanog结合结构的基因的调节区域中存在特异性的5hmC的富集现象。TET1和TET2同属于TET酶家族，并且在髓母细胞瘤中均有很高的表达水平，然而，只有敲除Tet1基因的SmoA1模型小鼠可以改善小脑肿瘤不受控制的增殖现象，并且改善小鼠的预后。药理学的Tet1抑制剂可以降低MB细胞活性以及FDGF信号通路相关基因的活性。因此，我们总结得出，在髓母细胞瘤的肿瘤发生过程中，5hmC起到重要作用，并且TET1具有致癌基因属性，可以作为治疗髓母细胞瘤的潜在治疗靶点。