循环微囊泡中miR-26a通过Wnt信号通路调控肝癌转移复发的机制研究

Hepatocellular carcinoma (HCC) is one of the highest malignant diseases. More than 70% of HCC patients lost the opportunities for radical resection. Even for patients who recived surgical resections, the 5-year recurrence rate was still over 75%. Therefore, there is an urgent need to investigate the mechanism of HCC metastasis. Recent studies reveal that the circulating microvesicles, containing microRNAs and proteins, are important mediators in the crosstalk between tumor cells and microenvironment. Such crosstalk mediated by microvesicles play essential roles in the proliferation, migration, chemotaxis and plant of tumor cells. In our previous studies, we obtained the expression profiles of microRNAs and proteins of circulating microvesicles from HCC patients via high throughput microarray techniques. We found that HCC patients with recurrence showed significantly lower expression of miR-26a and higher protein expression of Frizzled-5 in circulating microvesicles. Further target prediction recognizes that the target genes (WNT5A, APC, LEF1) of miR-26a are key regulatory factors in Wnt signaling pathway. Based on the recent discoveries and our new findings, this study aims to explore the mechanism of circulating microvesicle-mediated miR-26a for promoting HCC metastasis via Wnt signing pathway. In this research proposal, we will perform the in vitro and in vivo studes on transducting nucleic acid to microvesicles, co-immunoprecipitation and nude mouse HCC model intervention for investigating the possile mechanisms. The outcomes in this study will provide the scientific basis for screening tumor biomarkers and developing novel approaches for cancer target therapy.

肝癌恶性程度高，其转移复发机制的探索是目前研究的难点。新近研究表明，循环血中的游离微囊泡可作为微环境与肿瘤细胞交互对话的的载体在肿瘤细胞的趋化、运动、黏附、定植过程中发挥重要作用，并与肿瘤转移复发密切相关。我们前期利用高通量技术对肝癌患者外周血循环微囊泡内的microRNA、蛋白质进行表达谱差异分析，发现复发患者循环微囊泡中miR-26a显著下调而Wnt信号通路的重要受体Frizzled-5显著上调。靶基因预测提示miR-26a是Wnt通路WNT5A、APC、LEF1的调控因子。本课题将在前期研究基础上，通过微囊泡核酸转导、免疫共沉淀、裸鼠肝癌模型等体内外实验探究循环微囊泡中miR-26a 通过Wnt 信号通路调控肝癌转移复发的分子机理；并组装携带人工合成miR-26a模拟物的微囊泡进行裸鼠肝癌模型的干预实验。为寻找肝癌转移复发的分子标记物和新的靶向药物给药途径提供新思路和方向。

肝细胞癌（以下简称肝癌）恶性程度高，手术切除是首选的治疗方式，但术后的转移复发是影响生存的主要因素。复发转移机制的探索是目前研究的难点。本课题在前期研究基础上，将携载外源性miR-26a的微囊泡与肝癌细胞株 SMMC-7721 和 MHCC-LM3 共培养48小时后，肝癌细胞miR-26a表达水平相显著升高而其增殖、迁移、侵袭能力受到显著抑制。结合miRNA 靶基因预测数据库、靶基因验证和双荧光素酶报告基因检测发现，微囊泡中miR-26a 可通过直接调控WNT关键基因LEF1，下调其下游 CCND1、CCND2 和 CCNE1 表达， 从而抑制肝癌细胞增殖、迁移和侵袭能力。小动物活体成像系统检测发现，携载 miR-26a的微囊泡通过尾静脉注入动物体内后，主要积聚于肝脏，且持续时间长（48 小时）；肿瘤体积明显小于对照组（SMMC 7721 组，p <0.001； HCC LM3 组：p=0.002）；免疫组化检测发现，实验组的 SMMC 7721和 LM3成瘤组中 Ki-67，LEF1，Cyclin D1，Cyclin D2，Cyclin E1表达较对照组均显著下降。本研究提示微囊泡可运输抗肝癌活性物质 miR-26a进入体内并积聚于肝脏，通过WNT通路影响肝癌表型而抑制肝癌进展，从而发挥药物运输载体功能。