miR-759在原发性肝细胞癌恶性进展中的分子功能和网络调控机制及其潜在的治疗作用

It has been demonstrated that the development of Hepatocellular Carcinoma (HCC) has been accompanied by the multiple-molecular changes and the multiple-step complicated process. It has been reported that altered microRNA expression plays the important role in HCC aggressive progression. Recently, we found that the expression of miR-759 was significantly down-regulated in HCC cell lines and plasma of patient with metastasis. So maybe the low expression of miR-759 has associated with the invasion and metastasis of HCC . However, it has been unknown for the molecular carcinogenic character and relative regulating network mechanisms of miR-759 disregulation in HCC aggressive progression.. We will examine the expression of miR-759 in the tissue and plasma of normal and HCC patients by the use of associated molecular oncological study methods and high-output detection technology and analyze the correlation between the expression levels of miR-759 and HCC clinical pathological parameters. In addition, we will explore the effect of the overexpression of miR-759 in HCC cell lines on the tumor biological behaviour, such as the formation of stress fiber, filopodial, lamellipodial,directional movement,epithelial-mesenchymaltransition, migration,.invasion and tumor angiogenesis and in vivo tumor metastasis. All the more, we will study the direct target gene of miR-759 and the important down-stream associated-metastasis gene. Then we will find that the clinical oncological significance of miR-759 and the related target gene abnormity in HCC aggressive progression. And we will explore that the potential therapeutical effect of miR-759 on the tumor metastasis,life expectancy and the formation of the ascite in the HCC orthopedic pulmonary metastatic model. We will furtherly research the molecular mechanisms which result in the low expression of miR-759, and find a new signalling network pathway which regulates the expression of miR-759. So we will devote ourselves to supplying the experimental scientific support for the deep recogniztion of the molecular genetic character in HCC aggressive progression.

预实验发现，miR-759在HCC细胞系和血浆中表达明显下调，且与HCC的侵袭转移相关。但miR-759异常在HCC恶性进展中的分子致癌特征和相关的网络调节机制还不清楚。.本项目拟应用分子肿瘤学研究方法和高通量检测技术，检测正常人和HCC病人组织、血浆中的miR-759表达，分析miR-759表达水平与HCC转移的相关性；分别探讨miR-759对HCC细胞骨架、丝状伪足、板状伪足、直线运动、EMT和肿瘤血管形成、动物体内肿瘤转移等生物学行为的影响；确定HCC中miR-759作用的靶基因和重要的下游转移相关基因；明确miR-759作用的靶基因异常表达的信号转导通路及其在HCC恶性进展中的临床肿瘤学意义。然后采用动物转移瘤模型，探讨miR-759对HCC体内转移的潜在治疗效果；深入研究miR-759在HCC发生发展中表达失调的分子网络机制，致力为全面认识HCC的分子遗传学特征提供实验研究的依据

我们在实验中发现miR.-759、miR-449a在HCC细胞系和组织中均呈较低水平的表达，而在正常永生化的细胞株LO2 和HCC癌旁临近的非肿瘤性肝组织中呈高水平的表达。并且miR-101在有远处转移的HCC患者的血浆中的表达明显低于未转移的HCC患者。相关性分析显示：miR-759、miR-101、miR-449a低水平表达与HCC的恶性进展密切相关，而且是评估HCC患者预后的独立指标 (P<0.05)。进一步功能实验证实miR-759、miR-101、miR-449a在HCC细胞中的稳定过表达可显著抑制肿瘤细胞的体外增殖、迁移和浸润能力，并明显抑制HCC细胞在动物体内肝脏和肺脏的转移灶形成。另外，miR-759、miR-449a和miR-101在HCC细胞系的过表达明显逆转了细胞的EMT，而miR-759、miR-101抑制肿瘤细胞张力丝、板状伪足、丝状伪足的形成。同时在肝细胞癌肺转移的裸鼠模型中，将miR-101慢病毒液经裸鼠尾静脉注射治疗发现miR-101抑制了裸鼠肝脏、肺、纵膈的肿瘤转移而延长了裸鼠的生命并未见明显毒副作用。接着我们识别了MiR-759的启动子区，发现CTCF结合在miR-759的启动子区并激活miR-759的转录。并进一步识别了与CTCF连接的长非编码RNA lincRNA01137, 且lincRNA01137通过调节CTCF与miR-759启动子序列的结合而调节了miR-759的转录，从而揭示了lincRNA01137-CTCF-miR-759调节网络在肝细胞癌发生和恶性进展中的作用。