SARI基因在结肠癌中表达失活的机理研究

SARI gene is an important transcription factor and tumor suppressor gene. We found that SARI is a novel suppressor of tumor angiogenesis (Dai L, et al. Nature Communications. 2016), and low expression in human colon cancer. Further studies have demonstrated that SARI was down-regulated during AOM+DSS-induced colitis associated colon cancer. And DNA methylation-mediated SARI inactivation was found in various colon cancer cells. But, until now, the mechanism was still unclear and without literature reporting. In order to elucidate the mechanism of SARI gene inactivation in colon cancer and the effects of SARI demethylation specifically, sulfite treatment - methylation specific PCR-TA cloning are performed to detect the methylation site of SARI in 80-100 colon cancer tissues of patients with different clinical staging. The correlation between SARI methylation and the clinical staging and prognosis of patients were analyzed. Furthermore, dCas9-based demethylation technique was employed to remove the methylation of SARI gene in colon cancer cells specifically and investigate the effect of SARI demethylation on colon cancer growth. The present study would lay the foundation for understanding the mechanism of SARI inactivation in colon cancer and provide a potential therapy target for colon cancer.

SARI基因是一个重要的转录因子和抑癌基因，我们发现SARI是一个新的肿瘤血管生成抑制因子（Dai L, et al. Nature Communications. 2016），在人结肠癌中低表达。进一步研究发现SARI在AOM+DSS诱导的结肠“炎-癌”过程中表达下调，也在多种结肠癌细胞中存在DNA甲基化介导的表达失活，但机制不清楚，也未见文献报道。为了阐明SARI基因在结肠癌中表达失活机制及特异性去除SARI甲基化后的功能，本课题将利用亚硫酸盐处理-甲基化特异性PCR-TA克隆检测80-100例不同临床分期的人结肠癌组织中SARI基因的甲基化位点、分布情况以及与患者临床分期和预后的相关性；利用dCas9技术特异性解除结肠癌细胞中SARI基因的甲基化，研究特异性去除SARI甲基化后对结肠癌生长的调控作用，为阐明SARI基因在结肠癌中的失活机制奠定基础，也为未来结肠癌的治疗提供潜在的靶点。

经过课题组成员3年的研究，我们已完成了该课题的研究目标和任务，阐明了SARI基因在结直肠癌中表达失活的机理，获得了相关原创性研究成果。该课题的相关主要成果已经发表在Cancer Letters杂志（IF=7.36），该课题申请人为该论文的并列第一作者和并列通讯作者，该论文标注的第一基金号为该课题编号：81702349。依托该课题，共培养了1名博士研究生。.在该课题的资助下，我们首次在在结肠癌病人的肿瘤组织中阐明了SARI基因启动子区域的甲基化与其表达水平呈负相关，并且SARI基因启动子区的甲基化水平可以作为结肠癌患者的预后指标。在此基础上，我们构建的SARI基因特异性去甲基化的质粒dCas9TET1CD-sgSARI能精确地对SARI启动子进行定点、特异性去甲基化，从而内源性地激活SARI的表达，在结肠癌细胞株中内源性上调SARI基因的表达后能够促进结肠癌细胞的凋亡，抑制结肠癌细胞的增殖、迁移和侵袭能力，并抑制肿瘤的血管生成来共同发挥抗肿瘤作用。本课题研究将为未来结肠癌以及其他肿瘤的治疗提供了新的思路。