BTG2甲基化调控肝癌细胞放疗敏感性的分子机制研究

The status of DNA methylation and microRNA is associated with the radiotherapy sensitivity. BTG2 is a gene that could regulate key functional genes related to tumor chemoradiotherapy sensitivity. DNA methylation transferase (DNMTs) are target genes of miR-29b that has the ability of regulation on DNA methylation. Our previous study have demonstrated that BTG2 is a target gene of miR-29b, and both of them are down-regulated in liver cancer tissues and liver cancer cells. Methylation does not exist in BTG2 gene, however, the level of BTG2 expression increases and BTG2 gene has to be found to exist methylation in liver cancer cells after radiation. However, the relationship between methylation and tumor radiotherapy sensitivity has not been reported yet. Recent study has reported that BTG2 could regulate its translation function by binding with DNMTs. Thus, we propose hypothesis that miR-29b could regulate BTG2 directly by targeting BTG2-3’UTR or could induce BTG2 methylation to regulate radiotherapy sensitivity of liver cancer cell by DNMTs. From two points of epigenetics, we elaborate “The mechanism on the regulation of BTG2 methylation in HCC radiotherapy sensitivity” according to pyrosequencing, luciferase reporters, CHIP and WB etc, which will expand the new mechanism about the regulation of miR-29b to BTG2 and provide theory basis for explaining the mechanism of methylation in regulation of HCC radiosensitivity.

功能基因DNA甲基化状态和miRNA与肿瘤放疗敏感性密切相关。BTG2是调控肿瘤放疗敏感性的重要功能基因。我们前期证实BTG2是miR-29b的靶基因，miR-29b与BTG2在肝癌组织和细胞中都呈低表达，均未发现甲基化现象，而在肝癌细胞电离辐射后BTG2水平上调并发生高甲基化，而目前这种甲基化转换现象与miRNA和肝癌放疗敏感的相关性研究尚未见报道。miR-29b是DNMTs上游miRNA，可负向调节DNA甲基化；而近期报道DNMTs通过结合BTG2甲基化位点调节其转录功能。由此我们提出假设：miR-29b通过直接作用于BTG2或/和通过DNMTs介导BTG2甲基化调控肝癌细胞放疗敏感性。本课题拟采用焦磷酸测序、报告基因分析、CHIP和WB等方法旨在从表观遗传调控2个角度揭示BTG2甲基化调节肝癌放疗敏感性作用方式，拓展miR-29b调控BTG2的新机制，为诠释肝癌放疗抵抗提供新思路。

功能基因DNA甲基化状态和microRNAs与肿瘤放疗敏感性密切相关。BTG2是调控肿瘤放疗敏感性的重要功能基因。我们前期证实BTG2是miR-29b的靶基因，miR-29b与BTG2在肝癌组织和细胞中都呈低表达，均未发现甲基化现象，而在肝癌细胞电离辐射后BTG2水平上调并发生高甲基化。在此基础上，我们采用生物信息学分析、临床样本检测，联合地西他滨与放疗处理肝癌细胞，然后以流式细胞术、RT-PCR、WB、MSP等方法，研究miR29b、BTG2表达及其DNA甲基化之间的相互影响关系，以及BTG2甲基化对肝癌放疗敏感性的影响。本研究结果证实：肝癌临床样本中BTG2基因表达水平与预后相关；BTG2甲基化水平与肝癌患者预后相关；地西他滨去甲基化处理对BTG2表达有影响，地西他滨预处理会影响肝癌细胞的放疗敏感性。总之，本课题的研究是对前期工作的进一步拓展和延伸，把目前认为对肿瘤放疗敏感性有重要作用的基因BTG2、DNA甲基化以及mcroRNAs联系起来，不仅有助于进一步阐明BTG2基因放疗增敏的机制，同时也有助于探索肝癌放疗抵抗中相关信号通路的选择模式，为解决今后对不同基因状态的临床肝癌患者实施个体化的基因治疗提供理论和实验基础。