miR-124-3p靶向PRAS40在肝细胞癌发生中的作用与机制研究

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death worldwide. The incidence of HCC is increasing, whereas the treatment options are limited and often inefficient. Therefore the novel treatments are needed urgently. However, the molecular mechanism of HCC tumorigenesis remains largely unknown. PRAS40 is the substrate of Akt, and may play important roles in multiple tumors. We have reported the important role of PRAS40 in Ewing’s sarcoma, whereas the effects of PRAS40 on HCC need to be determined. Recently, our results showed that the protein level of PRAS40 was increased significantly in human HCC samples, and the high level of PRAS40 protein was correlated to a poor prognosis. Furthermore, PRAS40 depletion resulted in a suppression of cellular proliferation. These data suggested a very important role of PRAS40 in HCC tumorigenesis. We found that the tumor suppressor miR-124-3p could bind the 3’UTR of PRAS40 mRNA and suppress the translation of PRAS40 in luciferase assay. Therefore, we made a hypothesis as follows: The low expression of miR-124-3p led to an abnormally increased protein level of PRAS40, which could contribute to the tumorigenesis of HCC. .Based on the previous data, we will study the effects of PRAS40 on HCC tumorigenesis further by using the HCC clinical samples, cell lines and knockout mice. We will clarify the role of miR-124-3p in PRAS40 targeting and the correlation between the tumor suppressive role of miR-124-3p and PRAS40 expression. The aims of this project are to determine the role and the mechanism of high PRAS40 protein level in HCC tumorigenesis, which could be expected to offer novel clues for the HCC treatment.

肝细胞癌发生的分子机制尚不明确。PRAS40是Akt的作用底物，可能参与多种肿瘤形成。申请人曾报道其在尤文肉瘤中的重要作用，但其与肝细胞癌的关系不清。申请人近期研究表明PRAS40蛋白水平在人肝细胞癌中升高且与不良预后相关，体外沉默PRAS40抑制癌细胞增殖，提示其在肝细胞癌发生中很重要；并发现抑癌微小RNA miR-124-3p与PRAS40 3'UTR结合并抑制其翻译，故提出假说：miR-124-3p低表达使PRAS40蛋白水平失控而异常增高，从而促进肝细胞癌形成。本项目拟基于前期基础利用肝细胞癌临床标本、细胞系及基因敲除鼠模型，深入研究PRAS40对肝细胞癌发生的影响，揭示miR-124-3p与PRAS40的靶向关系，确认miR-124-3p的抑癌作用与PRAS40的相关性，阐明PRAS40在肝细胞癌发生中的作用及高表达的机制。以期为揭示肝细胞癌发生机制提供新线索，为治疗提供新思路。

原发性肝癌是肿瘤相关性死亡的第三大病因。肝细胞肝癌（HCC）占原发性肝癌的90%，恶性程度高,病情发展快,急需找到新的治疗方法。目前肝细胞肝癌的分子靶向治疗只有索拉非尼有效，远不能满足需求。PRAS40能够促进细胞存活、增殖和转移，抑制凋亡和老化。T246磷酸化型PRAS40在多种肿瘤标本中上调，但是PRAS40磷酸化水平的上调是基于其上游PI3K/Akt或mTOR通路的活化，还是其本身表达上调尚不得而知。另外，PRAS40在HCC形成中的作用也未见报道。.本项目中，我们分别在TCGA数据库和HCC临床标本组织中发现PRAS40和其磷酸化水平在HCC组织中均上调，且与患者生存期呈负相关。我们还利用DEN诱导小鼠形成HCC，发现与癌旁组织相比，肝癌组织中PRAS40的表达水平明显升高，编码PRAS40的基因AKT1S1敲除后，HCC形成受到抑制。在体外实验中发现PRAS40促进肝癌细胞增殖，抑制自噬介导的细胞死亡。并发现PRAS40敲低后Akt的磷酸化水平降低。在裸鼠移植瘤实验中发现在细胞中敲低PRAS40后，肝癌细胞成瘤性降低，而再次过表达PRAS40后，肝癌细胞的成瘤性部分恢复。.为了阐明PRAS40在HCC中表达上调的机制，我们预测了可能靶向PRAS40的小RNA。荧光素酶报告基因实验结果显示将miR-124-3p转入细胞后，含有PRAS40 3’UTR的荧光素酶表达水平明显低于对照组，而将PRAS40 3’UTR结合的碱基突变后其表达无明显变化；miR-124-3p抑制子则没有这种效果。将miR-124-3p转入肝癌细胞后，细胞增殖明显减弱，PRAS40表达水平明显降低；转染miR-124-3p抑制子后，细胞增殖和PRAS40蛋白表达水平均有上调。而过表达PRAS40则明显逆转了miR-124-3p对肝癌细胞增殖的抑制。另外，我们在HCC的临床标本和小鼠的肝癌组织中还发现miR-124-3p的表达与PRAS40和其磷酸化水平均呈负相关。.本项目阐述了PRAS40在HCC发生中的重要作用，揭示了miR-124-3p与PRAS40的靶向关系，为阐明HCC的发生机制提供了新线索。