MeCP2-miR-22-MTHFD2/MTHFR轴对胃癌表观调控机制的研究

Epigenetic regulation of genes is associated with the development of gastric cancer. We have previously found that MeCP2, MTHFD2 and MTHFR are over-expressed in gastric cancer, with miR-22 down-expressed; MeCP2 bind to the promoter region of miR-22 gene; and there have the binding sites in the 3'-UTR of MeCP2, MTHFD2/MTHFR with miR-22. Therefore, we propose that MeCP2-miR-22-MTHFD2/MTHFR axis plays an important role in the epigenetic regulation of gastric cancer. In the present study, firstly, the clinical significance of MeCP2, miR-22, and MTHFD2/MTHFR in gastric cancer is observed using immunohistochemical, immunoblotting, and quantitive real-time PCR analysis. Then ChIP-seq, ChIP-PCR, MS-PCR, and reporter assay are applied to explore the targeting mechanism of MeCP2 to miR-22, and miR-22 to MeCP2 and MTHFD2/MTHFR. Next, roles of miR-22, sh-MeCP2, sh-MTHFD2, and sh-MTHFR in the cell proliferation, cell cycle, apoptosis, invasion, and migration of gastric cancer cells are studied by MTT, colony forming experiment, and flow cytometry. Meanwhile, changes of SAMe, and total DNA methylation are analyzed by HPLC. Finally, the influences of MeCP2-miR-22-MTHFD2/MTHFR axis are explored in vivo using nude mice. We aim to explore the roles of MeCP2-miR-22-MTHFD2/MTHFR axis and the epigenetic regulation mechanism in gastric cancer development and progression, which will provide a new perspective on the pathogenesis and progression of gastric cancer.

胃癌的发生发展涉及表观遗传学改变。我们前期研究发现MeCP2、MTHFD2/MTHFR在胃癌组织中高表达，且miR-22低表达；MeCP2与miR-22启动子区结合，而miR-22靶向结合MeCP2、MTHFD2/MTHFR 3'-UTR区，提出MeCP2-miR-22-MTHFD2/MTHFR轴在胃癌表观调节中具有重要作用。本研究拟应用QT-PCR、免疫组化等方法分析MeCP2-miR-22-MTHFD2/MTHFR轴临床意义；ChIP-seq、MS-PCR、报告基因系统等研究MeCP2-miR-22-MTHFD2/MTHFR轴分子间的靶向关系；细胞生物学技术检测miR-22、MeCP2、MTHFD2/MTHFR等生物学作用；HPLC分析SAMe、DNA总甲基化变化；荷瘤裸鼠模型研究MeCP2-miR-22-MTHFD2/MTHFR轴在体效应，揭示其在胃癌发生发展中的作用及表观调控机制。

胃癌的发生发展是一种多因素参与、多基因改变的网络调控过程。近年研究表明，表观遗传学改变也是胃癌发生发展的重要原因，对胃癌的诊断和治疗具有重要意义。MeCP2是表观调控的关键性蛋白，MeCP2可通过与抑癌基因或原癌基因甲基化CpG位点结合表观调控其表达，进而影响肿瘤的发生发展。课题通过免疫组化、Western Blot、QT-PCR分析MeCP2、MTHFD2、MTHFR、miR-22与胃癌临床病理特征的关系，结果显示MeCP2在胃癌中表达显著上调并与G分期及肿瘤大小等临床病理特征相关，而与其它病理特征无相关性；miR-22在胃癌组织中表达显著下调，并于MeCP2表达呈负相关；MTHFD2和MTHFR表达上调，并于胃癌的G分期密切相关。应用染色质免疫共沉淀与高通量测序相结合的技术（ChIP-seq）、miRNA芯片分析、TCGA数据分析等探讨MeCP2结合的靶向基因的结合序列。结果显示MeCP2与miR-22在胃癌中的表达呈负相关，沉默MeCP2表达则促进miR-22表达，沉默CTCF表达则抑制miR-22表达，MeCP2与CTCF对miR-22表达存在拮抗性调控作用。进一步的研究证明上游增强子甲基化是调控miR-22表达的重要因素。CTCF在miR-22上游主导染色质构象的形成，而MeCP2拮抗CTCF的激活作用。. 通过生物信息学分析与MeCP2、MTHFD2、MTHFR相结合的miR-22结合位点。双荧光报告实验证明miR-22通过直接靶向MeCP2、MTHFD2和MTHFR抑制胃癌细胞增殖，诱导凋亡。MTHFD2与MTHFR均促进胃癌细胞增殖。过表达MeCP2、MTHFD2和MTHFR消除了miR-22对胃癌细胞的影响。构建稳定表达miR-22的慢病毒载体，建立稳定表达miR-22的胃癌细胞系。结果显示MiR-22可导致SAM缺陷，并通过抑制甲基化来促进肿瘤抑制基因的表达，进而抑制胃癌的生长。进一步的研究发现SAM对胃癌细胞生长的影响呈剂量依赖性，与miR-22、MTHFD2和MTHFR表达密切相关。通过体内实证明MeCP2-miR-22-MTHFD2/MTHFR轴在胃癌进展中发挥着重要的调控作用。揭示了其在胃癌发生发展中的作用及正反馈的双环路表观调控分子机制，为胃癌的治疗提供了新的思路及基因治疗靶点。

内容获取失败，请点击重试