产肠毒素脆弱拟杆菌激活CACClnc-KAT2A-RAD51通路促进结直肠癌化疗耐药的机制研究

Chemotherapy resistance has become an important risk factor threatening the survival of patients with advanced colorectal cancer. The intestinal microbiota may modulate local intestinal responses and in turn affect chemotherapy. Our team has confirmed that Fusobacterium nucleatum promotes chemoresistance to colorectal cancer by modulating autophagy. However, it is not clear whether other pathogenic bacteria, such as Enterotoxigenic Bacteroides fragili (ETBF), which is involved in the tumorgenesis of colorectal cancer (CRC), may participate in the chemotherapeutic process of CRC patients. According to our previous data, we have proved that ETBF may promote chemoresistance to CRC cells in vitro and in vivo conditions. It is possible that ETBF may up-regulate lncRNA CACClnc expression, which recruits KAT2A protein to the promoter regions of those genes, which is closely correlated with repair of DNA damage, and activates these genes expression. The activation of repair of DNA damage correlated genes will lead to the enhancement of DNA homologous recombination repair ability in CRC cells, and finally induce chemoresistance in CRC cells in response to Oxaliplatin treatment. Therefore, we will verify our hypothesis in clinical samples; animals and cell models by employing RNA pull down, ChIP seq bioinformatica analysis, gene editing and other methods. We will also establish whether and how manipulation of gut microbial dysbiosis may be used as a potential therapeutic approach in the treatment and block of human CRC chemoresistant progression.

肿瘤细胞耐药频繁发生已成为威胁中晚期结直肠癌患者生存质量的重要危险因素，肠道菌群可通过调节细胞局部反应，进而影响化疗效果。申请者前期证实具核梭杆菌可能通过激活自噬促进结直肠癌化疗耐药，但是产肠毒素脆弱拟杆菌（ETBF）对结直肠癌化疗的潜在影响未有相关研究。根据前期实验数据，申请者证实结直肠癌细胞感染ETBF后能抵抗化疗药物奥沙利铂诱导的细胞杀伤，其机制可能是ETBF上调lncRNA CACClnc的表达后，促进DNA损伤修复关键基因转录表达，增强肿瘤细胞DNA的损伤修复能力，最终导致结直肠癌细胞化疗耐药。因此我们将在临床标本、体内外实验中通过RNA pull down、ChIP seq生物信息分析、基因编辑等手段阐释上述网络关系，探讨ETBF诱导结直肠癌细胞化疗耐药的机制并寻找潜在干预靶点。这一研究可为我们深入理解肠道菌群参与结直肠癌耐药发生的机理及研发有效干预措施提供理论基础。

产肠毒素脆弱拟杆菌（Enterotoxigenic B. fragilis, ETBF）处理肠道细胞后产生的外泌体中miR-149-3p减少，进而促进Th17分化，加剧肠道“炎-癌”转化，揭示了肠道致病菌在“炎-癌”途径中发挥的潜在作用，为炎症性肠病患者的防癌策略提供新靶点。长链非编码RNA（lncRNAs）在肿瘤发生中起重要作用。然而，lncRNA对化疗耐药和RNA可变剪接（RNA alternative splicing, RNA AS）的影响仍不清楚。在本研究中，我们发现了一个新lncRNA，CACClnc，在结直肠癌（colorectal cancer, CRC）中上调并与化疗耐药和不良预后相关。CACClnc通过促进DNA修复和增强同源重组促进结直肠癌细胞对化疗的耐受性。机制上，CACClnc与YB1（Y-box-binding protein 1，一种剪接因子）和U2AF65（U2AF剪接因子的亚基）特异性结合，促进YB1和U2AF65之间的相互作用，然后调节RAD51 mRNA的可变剪接，从而改变结直肠癌细胞的生物学功能。在结直肠癌患者外周血清外泌体中CACClnc的含量可以有效预测患者的化疗效果。因此，检测与靶向CACClnc及其相关通路将对结直肠癌患者的临床管理具有指导作用，并可能改善结直肠癌患者的预后。