IGF2印记丢失诱导结直肠癌干细胞自噬促进肿瘤复发转移的机制及其临床应用研究

Insulin-like growth factor (IGF2) imprinted loss (LOI) is common in colorectal cancer (CRC) cells, which play an important role in the development of CRC tumor stem cells (CSCs) which are closely related to CRC recurrence and metastasis. Our previous study found that compared with CRC imprinted (MOI) tissues and cells, CRC LOI tissue and intracellular tumor stem cells were significantly enhanced in dryness and significantly increased autophagy after induction, but the mechanism of IGF2 LOI promoting CSCs production and autophagy still remains unclear. We predict that IGF2 LOI in CRC cells firstly promotes overexpression of IGF2, which binds to IR receptor after translation by binding with lncRNA 91H-HNRNPK in a large amount, then activates GSK3b, inhibits mTOR expression, and promotes CRC stem cells formation with autophagy which eventually leads to the recurrence of CRC. Therefore, based on the previous studies, this project intends to analyze the regulation mechanism of IGF2 LOI-induced CSCs autophagy and its relationship with the prognosis of CRC patients from the three aspects of cellular molecular mechanism, animal model and clinical validation, and further explore its valus as a marker of CRC recurrence and metastasis in efficacy evaluation and prognosis judgment.

结直肠癌（CRC）细胞中普遍存在胰岛素样生长因子（IGF2）印记丢失（LOI）现象，后者在CRC肿瘤干细胞（CSCs）发生发展中发挥重要促进作用，而CSCs与CRC复发转移密切相关。我们前期研究发现CRC IGF2 LOI组织与细胞内CSCs干性显著增强且诱导后细胞自噬显著增加，但IGF2 LOI如何促进CSCs产生自噬的机制仍不明确。我们预测：CRC细胞发生IGF2 LOI，促进IGF2 mRNA过表达，后者蛋白在lncRNA 91H-HNRNPK蛋白复合体调控下，大量结合胰岛素受体（IR），激活GSK3b而抑制mTOR表达，促进CRC干细胞形成后发生自噬，进而影响CRC复发转移。本项目拟在前期研究的基础上从细胞分子机制、动物模型和临床验证三个层面研究IGF2 LOI所致CSCs自噬的调控机制及其与CRC患者预后关系，进而探讨其作为CRC复发转移标志物在疗效与预后评估中的价值。

肿瘤干细胞（Cancer stem cell，CSCs）可来源于具有干性的肿瘤细胞，其与肿瘤复发转移密切相关。目前结直肠癌（CRC）细胞中普遍存在H19 印迹调控区域（ICR）甲基化异常所致的胰岛素样生长因子2（IGF2）印迹丢失（LOI）现象，其引起IGF2在肿瘤组织中表达增高在维持肿瘤干细胞（CSCs）干性过程中发挥重要作用。另有研究指出CRC CSC干性与自身自噬水平密切相关，而IGF2在多种肿瘤自噬过程中也发挥着重要促进作用。因此，本研究以IGF2 LOI所致IGF2高表达是否存在通过自噬而调控CSCs干性作用机制入手，检测并验证了IGF2 LOI与CRC CSCs干性的相关关系，前者主要通过促进CSCs自噬维持细胞干性。随后研究从细胞水平、动物模型和临床病例三个层面探讨IGF2 LOI促进CRC CSCs自噬进而影响细胞干性的具体机制，得到如下结论：本研究成功验证了IGF2 LOI可通过促进CRC CSCs自噬来维持细胞干性的调控作用。研究表明IGF2 LOI常引起IGF2高表达，后者可通过与高表达的IR-A结合促进AKT蛋白表达，阻断GSK3β/Bcl-2通路对CSCs 自噬的抑制作用，从而发挥对CSCs细胞干性维持作用。研究进一步探索了CRC IR-A蛋白高表达与CRC患者细胞内miRNA-195的降解密切相关。IGF2 H19 ICR区域的甲基化或可作为CRC复发转移一个筛查指标用于辅助临床诊断。