LRP16及其剪接体在肝癌中的表达和作用研究

Hepatocellular carcinoma (HCC) is one of the world's leading cancer threats, especially among Asian population. However, the mechanism of HCC invasion and metastasis remain largely unknown. LRP16 (Leukemia related protein 16) is an oncogene associated with sex hormones, and overexpressed in several types of tumors, including breast cancer and prostate cancer. Recently, we found that LRP16 showed lower expression in HCC samples compared with normal tissues, and the promoter of LRP16 showed significant methylation, which were contrast with previously reported. Moreover, for the first time we detected a LPR16 splice variant(LPR16s) in HCC samples, suggesting the special role of LRP16 in HCC. Thus, in the proposal, we will further determine the expression and location of LRP16 and splice variant, the level of methylation in HCC. Then, we investigate the relationship between LRP16 and tumor clinical pathology and patient prognosis. By using HCC cell lines, we will determine the effect of LRP16 and LRP16s on the growth and metatasis of HCC through a series of in vitro assays, including MTT, transwell migration, soft agar colony formation and wound-healing assay, and in vivo animal experiments. Furthermore, gene chip technology, and bioinformatics analysis will be performed to find downstream target molecules of LRP16 gene, and clarify the possible signal transduction mechanisms.Thus, our findings will not only clarify the role of LRP16 in HCC, might but also provide an candidate target or marker for HCC therapy and prognosis.

LRP16是本单位克隆的一个激素相关癌基因，其在激素相关肿瘤中高表达，与肿瘤进展密切关联。近期我们对肝癌组织标本检测发现LRP16基因表达反而下调，且其启动子甲基化状态升高与以往其它肿瘤研究中报道相反。在克隆该基因的过程中，我们首次发现人肝癌组织中LRP16基因有剪接体存在，提示LRP16可能在肝癌中具有独特的调控机制。本项目将通过对肝癌组织标本中LRP16及剪接体(LRP16s)的表达和细胞定位，以及甲基化水平进行检测，同时结合临床资料分析其与肝癌转移、预后、生存期等临床指标的相关性。利用肝癌细胞系过表达或干扰LRP16及 LRP16s，体外细胞生物学实验和体内活体荧光动物成像模型来研究LRP16在肝癌生长、侵袭、转移及EMT过程中的作用，并运用基因芯片、免疫共沉淀等技术研究其作用机制。本项目研究的意义不仅在于明确LRP16在肝癌侵袭转移中的作用，并可能为肝癌的诊治提供新的分子靶点。

LRP16是本单位克隆的一个激素相关癌基因，其在激素相关肿瘤中高表达，与肿瘤进展密切关联。在本课题中我们研究发现肝癌组织标本中LRP16基因表达反而下调，收集肝癌患者临床资料，研究发现LRP16表达水平与肝癌患者的分级及肝外转移呈负相关。进一步检测发现LRP16水平与肝癌组织中转移和EMT相关指标E-cadherin、N-cadherin和Vimentin呈负相关。在克隆该基因的过程中，我们首次发现人肝癌组织中LRP16基因有剪接体LRP16S存在。检测多株肝癌细胞系中LRP16mRNA水平，与正常肝细胞相比，大多数肝癌细胞系中LRP16也呈低水平表达。为了进一步研究LRP16在肝癌生长转移中的作用，在本年度中我们成功构建LRP16基因的表达载体、干扰载体，并转染肝癌细胞系，体外检测证实我们成功获得高表达LRP16和低表达LRP16的肝癌细胞系，用于后续体外和体内功能细胞学研究。根据一年期研究计划，我们已经超额完成本课题的研究内容，完成研究生培养1名，并开始撰写研究论文。