环状RNA circHIPK2靶向miR-124调控Eps8在慢性髓系白血病耐药中的作用及机制

Circular RNA is involved in post-transcription of microRNA on the target gene as a competitive endogenous RNA, but there was no reports about its effects on chronic myeloid leukemia. Our findings suggest that: (1) Eps8 is highly expressed in CML and mediates imatinib resistant via AKT/mTOR pathway. (2) circHIPK2 expression in imatinib-resistant cells was significantly higher than imatinib-sensitive ones, and negatively corretated with miR124, while positively correlated with Eps8 expression. (3) Overexpression of circHIPK2 caused a decrease of miR-124 expression. It increased proliferation and decreased apoptosis of CML cells. Thus, we hypothesize that circHIPK2 inhibits the binding of miR-124 to Eps8 mRNA 3'UTR by competing with miR-124,which regulates the expression of Eps8 and mediates CML cells resistance to imatinib. This topic will be clarified both in vitro and in vivo: circHIPK2 promotes CML biological activity by regulating miR124/Eps8/Akt axis. This study is expected to clarify that circHIPK2, miR124 and Eps8 mediate the functions of CML in the regulation of drug resistance and provide a new idea for the treatment of CML.

环状RNA作为竞争性内源RNA，参与miRNA对靶基因的转录后调控，但它在慢性髓系白血病中的作用尚未见相关报道。我们前期研究发现：①Eps8在CML中高表达，并通过AKT/mTOR介导对伊马替尼耐药。②伊马替尼耐药细胞中circHIPK2表达升高，与miR124表达负相关，与Eps8表达正相关。③过表达circHIPK2引起miR124表达下降，促进CML细胞增殖，抑制凋亡，沉默则相反。由此提出假说：circHIPK2与miR124相互竞争，抑制miR124与Eps8 mRA 3’UTR结合，调控EPS8表达，激活AKT/mTOR信号，进而介导CML耐药。为验证该假说，本课题拟从体外和体内两个层面明确：circHIPK2通过调节miR124/Eps8/AKT轴，增强CML活性。本研究旨在阐明circHIPK2、miR124、Eps8三者介导CML耐药的分子机制，为CML治疗提供新策略。

在后TKI时代，耐药是影响慢性髓系白血病患者长期生存的瓶颈。根据前期工作基础，我们提出假说：circHIPK2通过ceRNA机制调节miR-124的活性，miR-124通过作用于Eps8 mRNA 3’ UTR，调控Eps8的表达，引起Eps8下游Akt/mTOR通路活性增强，最终导致CML细胞活性增强和对伊马替尼耐药。首先，我们通过临床水平和细胞水平验证伊马替尼耐药CML细胞circHIPK2高表达，miR-124低表达，且其差异表达及与Eps8表达水平均相关；采用慢病毒沉默和过表达技术，在体外和动物水平证实circHIPK2促进CML增殖、抑制凋亡和对伊马替尼敏感性；而后的机制研究证实了circHIPK2和miR-124的直接作用及miR-124和Eps8的特异性结合；通过沉默、过表达技术及rescue实验，证实circHIPK2 竞争性结合 miR-124 调控 Eps8 及其下游 Akt/mTOR 通路增强CML细胞活性。此外课题组还证实了小分子化合物leonurine通过SOCS5/JAK2/STAT3增强CML细胞对伊马替尼敏感性。本研究有助于深入理解和认识CML耐药的机制，可望为克服CML耐药提供理论与实验依据。