HMGA1靶向Akt调控葡萄膜黑色素瘤生长及转移的分子机制

No treatment is found to be effective on metastatic uveal melanoma (MUM) till now. It is pivotal to screen such patients and find effective therapeutic interventions on the metastasis in the early stage. Our previous preliminary investigation suggested that HMGA1 protein was highly expressed in UM assays, and the expression of HMGA1 was positively correlated with proliferation, low differentiation, invasion and metastasis and short UM- specific survival. On the result of interference of the HMGA1 expression by siRNA techniques, the proliferation, migration and invasion of UM cells were significantly inhibited. MiR-222 binded with 3′-UTR of HMGA1 gene, possibly regulates the expression of HMGA1. Previous studies demonstrated that HMGA1 is expressed highly in the poor differeniated tumors, lowly or no in the adult tissues, which suggests that the interference on HMGA1 gene probably block the migration and invasion of tumor through PI3K-Akt-MMP9 pathway, little on the normal cells of host, which is potential anti-tumor and anti-metastatic strategy. Therefore, we propose a hypothesis, HMGA1 is the oncogene in the UM cells, regulates the growth, apoptosis, migration and invasion of UM cells through PI3K-Akt- MMP-9 pathway. Our future study will be focused on the regulatory mechanism of HMGA1 in UM, performed by transfection of adenovirus, Real-time PCR, Western blot, Luciferase reporter system, and immunochemistry on UM cell lines, animal models, UM assays and liver metastatic assays. The project is proposed to provide a potential candidate therapeutic intervention on metastatic UM by targeted HMGA1 gene in the early stage.

转移性葡萄膜黑色素瘤（MUM）目前无有效治疗手段，早期发现并控制UM转移是该疾病研究的关键领域。本课题组研究发现HMGA1蛋白在UM患眼中高表达，与UM增殖、侵袭、转移及生存时间缩短相关；经siRNA技术干扰HMGA1表达，UM细胞的生长、增殖、侵袭和迁移显著抑制；HMGA1可能是miR-222靶基因。HMGA1在人类低分化肿瘤内高表达，正常组织中低表达或缺如，因此，抑制HMGA1可能仅影响肿瘤，对宿主正常细胞影响小，有望取得靶向抗肿瘤及其转移的疗效。据此，我们提出假说：HMGA1是UM的癌基因，激活PI3K-Akt- MMP-9通路，可促使UM细胞增殖、侵袭和迁移。我们拟使用UM细胞株、动物模型以及UM患眼及肝转移癌标本，采用分子生物学、免疫组化等方法，探讨miR-222负向调节HMGA1，通过Akt抑制UM细胞的侵袭和迁移机制。本项目的研究结果将为临床早期控制MUM提供新的基因靶点。

HMGA1是葡萄膜黑色素瘤（UM）发生及进展过程中的重要致癌基因，其过表达状态与UM细胞增殖、迁移等功能密切相关。申请人前期研究结果证实，HMGA1的异常表达与UM患者肿瘤远处转移、高复发率等临床恶性表型密切相关；经siRNA干扰下调HMGA1表达后，UM细胞的生长、增殖、侵袭和迁移功能受到显著抑制。本项目在前期工作的基础上，通过慢病毒分别转染两种不同的UM细胞系C918和MUM-2B，从而下调细胞中HMGA1的表达，结果证实，HMGA1可通过激活PI3K/Akt/MMP9通路而加速肿瘤的增殖转移。我们查询了生物学网站miRTarBase进行了分子靶点预测，提示miRNA-222与HMGA1有相互作用靶点。利用miR-222-3p mimics及miR-222-3p inhibitor分别转染C918和MUM-2B细胞，结果显示在miR-222-3p mimics组中，C918和MUM-2B的增殖活性明显升高，凋亡程度降低且迁移能力增强；相反，在miR-222-3p inhibitor组中C918和MUM-2B的凋亡程度加重，细胞活性和迁移能力减弱。HMGA1表达水平与miR-222 mimics呈正向调控关系，与miR-222 inhibitor呈反向调控关系。RT-PCT及Western blot检测分析miR-222对PI3K/Akt/MMP9通路信号表达的影响。结果发现miR-222受到HMGA1基因的直接调控，HMGA1可通过提高miR-222表达，从而激活PI3K/Akt/MMP9通路，促进UM肿瘤的发生和进展。最后，我们在裸鼠体内进行了再次验证，发现HMGA1可在体内发挥促瘤体增殖的功能，且此功能是通过miR-222调节作用和PI3K/Akt/MMP9通路共同参与完成的。故本课题通过抑制HMGA1表达，靶向性阻断MMP9上游的PI3K/Akt通路，从而阻止由MMP9介导的UM细胞侵袭和迁移，为临床UM肿瘤标志物和治疗靶点提供了可靠的基础数据和理论支持。项目资助发表SCI论文2篇；培养博士研究生1名，硕士研究生5名；举办国内学术会议2次；在北京参加第九届中国眼科学和视觉科学研究大会(CCRVO 2017)做大会发言并获大会奖学金。