A/U富集RNA片段通过Caspase 3 - PKCε途径调控肝癌细胞周期G2/M期的分子机制研究

C/EBPβ 3’UTR RNA can be independently in eukaryotic cell, and plays a vital role in cell normal physiological activities and metabolism. Recent research results of our group on tumor suppression of this 3’UTR RNA showed that its function was correlated to A/U-rich RNA fragment near to 3' terminus. To uncover how this A/U-rich RNA fragment functions as a tumor suppressive element, technologies, such as vector construction, RNA transfection and labeling, quantitative real-time PCR (qPCR), western blot, chromatin immunoprecipitation assay (ChIP), co-immunoprecipitation assay (Co-IP), mass spectrometry analysis (MS), laser scanning confocal microscopy (LSCM), flow cytometry (FCM), stable transfection cell screening, protein purification, phosphorylation analysis, PKCε protein kinase activity analysis, and cell cycle synchronization, will be explored to uncover these mechanisms and signal transduction processes: how this A/U-rich RNA fragment induces and activates Caspase 3, and how the activated Caspase 3 cleaves the whole PKCε into truncated PKCε (tPKCε, molecular weight is about 54 KD ), and then how tPKCε induces and maintains CK18 phosphorylation high and, as a result, to arrest cell cycle period at G2/M phase in hepatocellar carcinoma cells transfected with C/EBPβ 3’UTR A/U-rich RNA fragment. Moreover, human fresh hepatocarcinoma tissue is implanted in the nude mouse subcutaneously, and then the A/U-rich RNA fragment is injected into the transplanted nude mouse through caudal vein to verify the fact that C/EBPβ 3’UTR A/U-rich RNA fragment also functions as a tumor suppressive element in vivo. By this project, it establishes a theoretical basis and applies the animal evidence for tumor therapy with tumor suppressive element of nucleic acid.

C/EBPβ 3’UTR RNA可独立存在于真核细胞中并维持细胞正常生理活动和代谢。本组前期研究表明该3’UTR RNA 的肿瘤抑制功能与其3'端的A/U富集序列有关。为研究这段序列是如何发挥肿瘤抑制功能的，我们利用载体构建、RNA体外转录与标记、qPCR、western blot、ChIP、Co-IP、MS、LSCM、FCM、细胞筛选、蛋白纯化、磷酸化分析、酶活性分析、细胞周期同步化等方法对转染C/EBPβ 3’UTR A/U富集RNA片段的肝癌细胞进行分析，探索该RNA片段诱导和活化Caspase 3、剪切全长PKCε生成截短的 tPKCε、诱导和维持CK18高度磷酸化和导致肝癌细胞周期阻滞于G2/M期的分子机制和信号传导过程；并通过人肝癌组织裸鼠移植和尾静脉注射A/U富集RNA片段，证实A/U富集RNA片段在体内也发挥肿瘤抑制作用，为抑癌核酸元件的肿瘤治疗奠定理论基础和提供动物证据。

肝癌是一种死亡率极高的肿瘤，探索有效临床治疗手段和搜寻早期筛选标志物已刻不容缓，本课题从这两方面出发，对肝癌抑癌基因及其基因治疗可能性进行研究。本项目主要研究了A/U-rich RNA通过“海绵”吸附RNA结合蛋白HuR，减少HuR与Cyclin A2 mRNA的3'UTR RNA结合，降低Cyclin A2 mRNA稳定性，从而减少Cyclin A2蛋白表达，使肝癌细胞周期阻滞在G2/M期。在这一过程中，本组探究了PKCε/CK18和PXN对肝癌细胞的影响；同时还对HuR对肝癌转录组影响进行测序，发现HuR可以调控包括膜受体Stra6在内的基因表达，该部分后续内容正在深入研究中。在研究A/U-rich RNA功能时，本组发现了一种表达于肝细胞的新抑癌基因Shisa like 1 （SSL1），它属于Shisa超家族，定位于细胞高尔基体上，通过抑制Wnt5a和FZD5糖基化而影响蛋白成熟和分泌（膜定位），从而抑制非经典Wnt信号通路FZD5/Fyn/Stat3轴，降低多种癌基因表达和抑制细胞EMT进程，最终抑制肝癌增殖、迁移和侵袭。此外，本组还研究了细菌来源的RNase E截短体（N端，REt）对肝癌细胞的影响，发现REt可以抑制肝癌细胞体外增殖、迁移和侵袭，影响裸鼠体内成瘤等。上述结果证明A/U-rich RNA和REt将来可以用于肿瘤基因治疗，SSL1具有作为临床肿瘤筛选标志物和预后因子的潜力。