lncRNA-SLC25A25-AS1介导直肠癌放疗抵抗的作用机制研究

Neoadjuvant radiotherapy for rectal carcinoma has been demonstrated to significantly increase radical resection rate and improve prognosis. However, radioresistance is the main reason that causes the failure of radiotherapy. Although it has been confirmed that signal pathways of cell cycle, cell apoptosis, DNA damage, are key factors on radioresistance, their upstream regulatory mechanism remains unknown. Long noncoding RNAs (lncRNA) play an important role in epigenetics that they can regulate gene transcription through epigenetic modification. We have found that lncRNA-SLC25A25-AS1 can mediate radiation response by regulating the transcription of radiation response-related genes. And the level of SLC25A25-AS1 is much higher in tumor tissues of rectal cancer patients who are resistant to radiotherapy than in those of patients who are sensitive to radiotherapy. Based on this, we raise the hypothesis that SLC25A25-AS1 is an upstream regulator, who works by regulating the transcription of BUB1, thus mediates radioresistance of rectal carcinoma through pathways associated with irradiation response. In this study, we will further verify the effect of SLC25A25-AS1 on redioresistance of rectal carcinoma and its regulated genes using loss- and gain-of-function approaches combined with comprehensive evaluation of rectal carcinoma samples from patients who received neoadjuvant radiotherapy. And next we will illuminate in which way does SLC25A25-AS1 regulate gene expression through DNA methylation and modifications of histone detection. The goal of this study is to reveal the mechanism of lncRNA-SLC25A25-AS1 on rectal carcinoma radioresistance.

术前放疗可显著改善直肠癌患者预后，放疗抵抗是导致临床放疗失败的主要原因。目前虽已明确DNA损伤修复、细胞周期等相关通路是产生放疗抵抗的关键，但它们的上游调控机制尚不明确。lncRNA在表观遗传中起关键作用，能够调控基因表达。本课题组在前期工作中发现lncRNA-SLC25A25-AS1能够通过调控辐射反应相关基因的表达而介导直肠癌细胞放疗抵抗，因此提出假设：SLC25A25-AS1是直肠癌放疗抵抗中的上游调控基因，通过调节BUB1等基因表达，经由与辐射反应相关的作用通路而介导放疗抵抗。本项目将通过功能丧失／获得的实验方法，结合大量临床样本，进一步证实SLC25A25-AS1在放疗抵抗中的功能及其对BUB1的调控作用；采用DNA甲基化和组蛋白修饰检测明确其调控基因表达的方式，阐明SLC25A25-AS1介导直肠癌放疗抵抗的作用机制，为直肠癌放疗抵抗提供新的预测标志物及治疗靶点。

局部进展期低位直肠癌患者需通过术前新辅助放疗同步化疗增敏，从而达到提高保肛率，减少局部复发的治疗目标。然而，占比约70%的原发性放疗抵抗人群，是困扰临床实践的重大难题。项目负责人所在的课题组前期已成功构建两株获得性放疗抵抗大肠癌细胞株（RKO/RKO-R和HCT116/HCT116-R），利用肠镜下取活检组织样本构建PDX动物模型。为深入探究表观遗传学领域的调控机制，我们利用LncRNA芯片技术，为直肠癌放疗抵抗筛选新靶标。我们的研究工作主要包括四大部分：1）建立获得性放疗抵抗细胞株，利用LncRNA芯片筛选出与放疗敏感性相关的lncRNA，通过生信分析确定lncRNA SLC25A25-AS1在转录水平与放疗抵抗呈正相关，且下游信号通路主要富集在DNA损伤修复相关基因集；2）采用慢病毒转染方法，在放疗抵抗细胞株HT29和SW480中，分别敲低lncRNA SLC25A25-AS1表达水平，6Gy辐照剂量检测细胞增殖和克隆形成，结果显示lncRNA SLC25A25-AS1水平下调时，放疗敏感性明显增强，G1期细胞减少，S期细胞增多，细胞周期DNA合成抑制；3）转录组测序提示，随着lncRNA SLC25A25-AS1水平下调，表达下降最显著基因有BUB1、TRIB1、UBE2C、TPX2、TIMP1等。利用Pull Down技术筛选，GEO数据库验证，确认金属蛋白酶组织抑制因子1（TIMP1）是重要靶基因；4）我们还另外筛选出差异表达的miRNA，为进一步探究RNA层面的复杂调控机制，重点选取miR-423-5p验证其与放疗抵抗的相关性，并证实下调miRNA-423-5p可介导BcL-xL细胞凋亡通路促进放疗抵抗。以上重要研究成果，已分别整理成论文《TIMP1 Is A Potential Key Gene Associated With The Pathogenesis And Prognosis Of Ulcerative Colitis-Associated Colorectal Cancer》、《Downregulation of miR-423-5p contributes to the radioresistance in colorectal cancer cells》发表见刊。能精准预测放疗疗效的胚系突变基因模型，一定是极具临床前景的研究方向。