HMGA1介导lncRNA-LINC01615增强THBS2表达及其在肝癌转移中的作用与机制研究

The mechanism of long-chain non-coding RNA LINC01615 in hepatocellular carcinoma (HCC) metastasis is unclear. Applicant's previous study found that LINC01615 could enhance the expression of THBS2 with the help of HMGA1 protein. Overexpression of THBS2 could activate TGF-β pathway to activate downstream metastasis-related genes and promote HCC metastasis. Based on this, we speculate that the LINC01615/HMGA1/THBS2/TGF-β pathway plays a key regulatory role in HCC metastasis. The following research should be carried out: confirm the role of LINC01615 in HCC metastasis in vitro and in vivo, and focus on the molecular mechanism of the composition of LINC01615-HMGA1 complex promoting the formation of chromatin loops by promoters and enhancers, which in turn enhance THBS2 expression, as well as THBS2 activating downstream genes via TGF-β pathway; Transgenic mice are used to verify the function and molecular mechanism of LINC01615; Analyze the correlation between LINC01615 and its downstream genes and clinicopathological features of HCC. The purpose of this study is to explore new mechanisms of HCC metastasis and to provide new proof for finding more specific metastatic monitoring methods and treatment targets.

长链非编码RNA LINC01615在肝细胞癌（HCC）转移中的作用机制尚不明确。申请者前期研究发现LINC01615在HMGA1蛋白辅助下，能够增强THBS2的表达；过表达的THBS2能够激活TGF-β通路活化下游转移相关基因从而促进HCC转移。基于此，我们推测LINC01615/HMGA1/THBS2/TGF-β通路在HCC转移过程中发挥关键的调控作用。本研究拟通过：体内外实验确认LINC01615在HCC转移中的作用；重点研究LINC01615-HMGA1组成复合体并促使启动子和增强子形成染色质环，继而增强THBS2表达，以及THBS2通过TGF-β通路活化下游基因的分子机制；转基因小鼠验证LINC01615功能及分子机制；分析LINC01615及其下游基因与HCC临床病理特征之间的相关性。本研究旨在探索HCC转移新机制，为寻找HCC更具特异性的转移监测方法和治疗靶点提供新的依据。

转移是导致HCC治疗失败和患者死亡的最主要原因，然而HCC发生转移的机制尚不明确。研究证实，非编码RNAs调控在HCC转移过程中发挥了重要作用。我们发现长链非编码RNA LINC01615在转移HCC组织中表达上调，提示LINC01615可能参与了HCC转移。通过transwell和划痕实验证实，上调的LINC01615显著增强了肝癌细胞的侵袭和迁移能力。通过RNA免疫共沉淀、RNA-pull down结合qRT-PCR检测发现LINC01615能够与HMGA1形成复合体，二者结合后增强了THBS2表达水平。上调LINC01615后TGF-β通路分子表达升高，增强Twist蛋白表达，抑制E-cadherin转录，EMT进展增强；然而，沉默LINC01615后，上述指标水平变化则与之相反。沉默THBS2，显著逆转了LINC01615过表达所引起的TGF-β通路和EMT等分子的变化。加入TGF-β通路抑制剂SB431542，LINC01615或THBS2上调引起的促进EMT进程作用被中断。证实了LINC01615/THBS2通过TGF-β通路促进肝癌转移。Transwell实验发现，在肝癌细胞中沉默THBS2能够显著逆转LINC01615上调引起的肿瘤侵袭转移。沉默LINC01615可以显著缓解裸鼠尾静脉注射引起的肺转移。在LINC01615过表达的基因小鼠中，TGF-β通路分子升高，E-cadherin显著降低，TGF-β通路和EMT进程被活化增强，小鼠生存时间显著缩短。通过体内、体外实验，证实LINC01615/THBS2通过激活特定信号传导途径来调控肝癌细胞的侵袭和迁移特性。通过本项目研究，对LINC01615与HCC转移的关系进行了深入探究，丰富了肿瘤转移机制，为寻找HCC更具特异性的转移监测方法和治疗靶点提供了新依据。项目共发表学术论文3篇，培养硕士研究生3名。