长链非编码RNA LINC01296调控ASPP2表达促进肝细胞肝癌转移的机制研究

Metastasis is the important reason for death in hepatocellular carcinoma(HCC) and the mechanism of which is still not well documented. Long noncoding RNA(lncRNA) might have critical functions in cancer metastasis. We applied bioinformatics analysis and qPCR to detect LINC01296 expression in tumoral tissues and paired nontumoral tissues from HCC patients. LINC01296 expression was up-regulated in HCC tissues and higher expression of LINC01296 was significantly correlated with tumor size, BCLC stage and metastasis. Moreover, we found that knockdown of LINC01296 could inhibit HCC cells migration and invasion in vitro. We also found that LINC01296 could bind with PRC2 and epigenetically repress ASPP2 transcription in HCC cells. Bioinformatics analysis showed that LINC01296 promoter region contained two E2F1 binding sites. LINC01296 was downregulated by 3.4 folds while inhibition of E2F1 in HCC cells. Accordingly, the hypothesis is proposed:E2F1 promotes LINC01296 expression, and LINC01296 represses ASPP2 expression by binding with PRC2 and recruiting it to ASPP2 promoter region. We will document this hypothesis by clinical samples, RIP, ChIP, luciferase reporter assay and animal models et.al methods. Our work will further the understanding about the molecular mechanisms of HCC metastasis and provide a new therapy target for HCC.

转移为肝细胞肝癌患者死亡的重要原因且其分子机制仍未完全阐明。表观遗传学的改变如lncRNA参与了肿瘤转移的过程。课题组发现LINC01296在肝癌中表达显著上调，其表达与肿瘤大小、分期及转移呈显著正相关；干扰LINC01296后肝癌细胞侵袭转移能力明显下降，并上调ASPP2表达；RIP实验证实LINC01296与PRC2结合，ChIP实验证实PRC2与ASPP2启动子区结合。生物信息学发现LINC01296启动子区有2个E2F1结合位点，干扰E2F1后其表达下调3.4倍。据此提出假说：肝癌中E2F1促进LINC01296表达，LINC01296通过绑定PRC2介导ASPP2启动子区H3K27三甲基化抑制其转录，促进肝癌转移。本课题将通过临床样本验证，运用RIP、ChIP、荧光素酶报告基因及动物模型等实验证实上述假设，丰富肝癌转移机制，为肝癌的治疗提供新的靶点。

首先，我们在肝癌术后患者标本中验证LINC01296的表达，结果表明，LINC01296在癌组织中表达水平较癌旁升高，其差异表达情况与TCGA数据库结果以及我们的前期结果相一致，进一步的分析发现，LINC01296的表达与肿瘤大小、疾病分期相关。随后，通过细胞实验表明，LINC01296在肝癌细胞株中表达升高，敲低LINC01296可以抑制肝癌细胞侵袭转移并能够诱导ASPP2的表达上调。核质分离实验表明，LINC01296主要表达于细胞核内，并可以同时绑定PRC2的两个核心蛋白EZH2和SUZ12。敲低LINC01296或敲低EZH2后，ASPP2的mRNA和蛋白的表达均出现了上调..我们的研究证实，在肝癌的发生发展中，LINC01296表达升高，通过募集绑定EZH2蛋白，介导了下游基因ASPP2的转录沉默，从而导致肝癌细胞的侵袭转移。本研究首次阐明LINC01296在肝癌发生发展中的作用机制，基本完成了原定目标。在此青年基金的资助下，本人发表SCI论文2篇，其中共同通讯作者1篇，共同第一作者1篇，正在撰写1篇。协助培养研究生1名。