TPM2基因高甲基化激活RhoA在结直肠癌浸润转移中的作用及机制研究

Downregulation of TPM2 expression in colorectal cancers (CRC) has been identified and validated in our previous studies. However, the machanisms responsible for the downregulation of TPM2 remain unknow. Recently, by using the Illumina HumanMethylation450 Infinium array, we identified 24 genes which are differentially methylated in CRC as compared to match normal samples, including TPM2. The methylation of TPM2 is also found to be associated with the activation of RhoA, tumor invasion and metastasis. Therefore, we hypothesized that the downregulatioin of TPM2 might attribute to the methylation and promotes tumor invasiveness and metastasis via the activation of RhoA. In the present study, we apply the methylation-specific PCR, sodium bisulfite sequencing and Pyrosequencing to TPM2 gene. qRT-CPR or tissue microarrys will be used to detect the mRNA and protein expression levels of TPM2. The correlation among the methylation, mRNA and protein expression will be studied comprehensively. Further, the functions of TPM2 gene, including the suppression of RhoA activation, tumor invasivenss and metastasis, will be investigated on colon cell lines with altered TPM2 expression by using TPM2-specific siRNA knockdown or adenovirus-infected TPM2-transfection in vitro, and assessed by using a in vivo SCID mice model intensively. The mechanisms of downregultion of RhoA induced by TPM2 will be explored by using the single-allel-mutated RhoA construct in the promotor region and by luciferease assays. These findings will provide the foundamental evidences for the machnisms of TPM2 in the setting of the epigenectic target therapry in CRC.

我们已经证明TPM2基因在结直肠癌中表达下降。那么引起TPM2表达下调的机制是什么呢？采用甲基化基因芯片技术，我们发现了24个与结直肠癌相关的基因，其中包括TPM2；进一步实验显示：TPM2甲基化与RhoA激活及肿瘤浸润转移相关。因此提出"TPM2甲基化抑制其蛋白表达并激活RhoA促进结直肠癌的浸润转移"的科学假设。本项目拟采用甲基化特异性PCR、RT-PCR和免疫组化分别检测结肠癌中TPM2的甲基化状态、mRNA和蛋白表达规律并分析它之间的相关性；采用siRNA和腺病毒表达技术改变结肠癌中TPM2表达，经体外细胞实验和体内动物活体成像模型，观察TPM2对RhoA激活及肿瘤生长转移的影响，从"细胞-动物-病人"多个层次揭示TPM2在结直肠癌中的作用机制；并采用启动子点突变、荧光素酶分析探讨TPM2调节RhoA的具体机制。该研究的完成将对阐明结直肠癌浸润转移机制和发现新的治疗靶标提供依据。

我们在前期的研究中发现TPM2在结直肠癌中表达下调，因此本课题探讨了结直肠癌中TPM2下调的机制和潜在生物学效应。我们通过qRT-PCR和免疫组化分析结直肠癌中TPM2的表达，然后在TPM2过表达和低表达的细胞株中都分析了其生物学功能。启动子的异常甲基化是原发结直肠癌组织TPM2表达下调的一个关键因素，我们通过5-氮胞苷（5-AZA）处理结直肠癌细胞株，使其去甲基化后能导致TPM2表达上调。我们在细胞株中还发现， TPM2重构后可以抑制CRC细胞的增殖与转移，而TPM2的沉默则可以促进CRC细胞增殖与转移，这个过程伴随着RhoA的激活。因此，我们的研究主要发现在结肠肿瘤中， TPM2大部分都由于异常甲基化而不表达或低表达，而这个过程与RhoA的激活及肿瘤增殖密切相关。