肝癌侵袭转移中UC001kfo对α-SMA分子调控机制的研究

The invasion and matastasis of hepatocellular carcinoma (HCC) is the primary cause for the patients’ poor prognosis. The alpha smooth muscle actin (α-SMA) plays an important role in the invasion and metastasis of cancer cells, which is regulated by a variety of signaling pathways and proteins. In our initial experiments, a new long noncoding RNA (lncRNA) UC001kfo was found to promote the invasion and metastasis of HCC. lncRNA is a noncoding RNA with important gene regulations. Moreover, our previous and preliminary results demonstrated that the gene of α-SMA is being the target gene of UC001kfo, and UC001kfo promotes the invasion and metastasis of HCC through α-SMA. UC001kfo binding protein (UC001kfo-pro) is a key protein molecule of UC001kfo regulation function. The current project studies how UC001kfo regulates and controls α-SMA and promotes the invasion and metastasis of HCC. By techniques such as in vitro transcription of UC001kfo, the RNA pull down experiment, mass spectrographic analysis, RNA immunoprecitation (RIP), RT-PCR, western blot, UC001kfo-pro is identified and verified. By the cell model, gene expression and interference technique was used to study of UC001kfo regulating α –SMA through UC001kfo-pro. By cell and animal models, the important role of UC001kfo-pro for UC001kfo’s gene regulation in invasion and metastasis of HCC. This project illustrates UC001kfo’ mechanisms in the control and regulation of α –SMA in the invasion of HCC so as to further provides effective target points for the treatment of HCC.

侵袭转移是肝癌患者预后极差的主要原因。肝癌侵袭转移中，发挥关键作用的α-平滑肌肌动蛋白（α-SMA）受多种信号通路的调控，对其干预效果差。我们前期实验中发现一个新长链非编RNA UC001kfo通过直接调控α-SMA的表达，促进肝癌侵袭转移。UC001kfo的结合蛋白（UC001kfo-pro）是UC001kfo发挥调控功能的关键分子。本课题应用体外转录UC001kfo，应用RNA pulldown、质谱分析、RNA免疫沉淀分析、RT-PCR和western blot，鉴定和验证UC001kfo-pro；利用基因过表达和干扰技术研究UC001kfo通过UC001kfo-pro调控α-SMA的表达；研究UC001kfo-pro对UC001kfo发挥促进肝癌侵袭转移的意义。本课题阐明UC001kfo在肝癌侵袭中对α-SMA的调控机制，进一步揭示肝癌侵袭转移的机制，为肝癌治疗提供新靶点。

侵袭转移是肝癌患者预后极差的主要原因。肝癌侵袭转移中，发挥关键作用的α-平滑肌肌动蛋白（α-SMA）受多种信号通路的调控，对其干预效果差。我们前期实验中发现一个新的长链非编码RNA UC001kfo通过调控α-SMA的表达，促进肝癌侵袭转移。因而，本研究将以UC001kfo通过α-SMA调控肝癌转移侵袭的作用机制为主要目标，为以α-SMA为核心的肝癌转移侵袭调控通路的机制研究奠定坚实的基础。我们使用信息学预测核转录因子Sp1可以与α-SMA启动子结合，我们通过荧光素酶报告基因以及CHIP-PCR实验进行了验证和证实。UC001kfo通过与Sp1的泛素化位点结合从而抑制其泛素化。Sp1在多种肿瘤组织中呈高表达，Sp1通过调控下游基因的转录表达，参与调节肿瘤的生长、侵袭及血管生成等多种生物学行为。一条新的肝癌调控通路UC001kfo/Sp1/a-SMA呈现出来，即肝癌组织及细胞中过量的UC001kfo通过上调Sp1的表达，Sp1含量的增加直接上调受其调控的下游的基因a-SMA的转录，最终肝癌转移侵袭增强。我们对通路特异性及作用主导性做了分析，表明UC001kfo/Sp1/a-SMA主导了UC001kfo促进肝癌侵袭转移的过程。本研就的重要意义在于，我们发现并明确了一条新的肝癌侵袭转移调控机制UC001kfo/Sp1/a-SMA，这为LncRNA在肝癌肝癌侵袭转移中的作用机制的完善，以及以UC001kfo为靶点的肝癌基因治疗的前瞻性研究提供了理论基础。