脑硫脂通过miR-223调节整合素αV亚基的表达的研究

Our previous results showed that sulfatide, a glycolipid that is synthesized from cerebroside by the enzyme of CST (cerebroside sulfotransferase) is able to regulate integrin αV subunit gene expression in hepatocellular carcinoma cells (HCC), while miR-223 down regulates integrin αV. However the relationship between sulfatide and miR-223 remains elusive. Therefore, in this project, we will first investigate the expression of miR-223 and integrin αV in the samples from patients with hepatocellular carcinoma and analyze the correlation between them. The effect of miR-223 overexpression and silence on the cell migration and adhesion to fibronectin and vitronectin will then be determined in hepatocellular carcinoma cells, with the rescue assay by integrin αV knockdown or overexpression. Next we will determine and confirm the relationship between sulfatide and miR-223 expression. The cell modes with overexpression of sulfatide will be established. The effects of both endogenous sulfatide produced by CST transfection and CST knockdown, and exogenous sulfatide treatment on the expression of miR-223 will be investigated. After confirming the regulatory effect of sulfatide on miR-223, we will explore the molecular mechanism for sulfatide regulation. To do this, SMMC-7721 and BEL-7404 HCC cells were transfected with CST construct or being treated with exogenous sulfatide, and the occupancy of acetylated histone H3k9/14 and transcription factor C/EBPα will be measured on the promoter of miR-223 precursor gene by chromosome immunoprecipitation assay. We then investigate the influence of sulfatide on the acetylation of histone H3k9/k14 and the transcription factor C/EBPα in hepatocarcinoma cells. To further uncover the mechanism, we then analyze the recruitment of histone acetyltransferase (HBO1,MOZ) and histone deacetylase (HDAC1) on miR-223 promoter, and analyze the effect of sulfatide on the recruitment of HBO1,MOZ and HDAC1 on miR-223 gene promoter. To test the in vivo function and regulation, SMMC-7721 and BEL-7404 HCC cells will be transfected with lentivirus containing miR-223 or CST constructs. After weeks of HCC cell implanting and tail vein injection of nude mice, the metastasis will be evaluated. The expression of miR-223 and integrin αV subunit will determined in the lung and liver metastatic foci. Their correlation with sulfatide is then analyzed and reconfirmed. In this project, we investigate the effect of sulfatide on miR-223 transcription and demonstrate the regulatory importance in the migration and metastasis of hepatocellular carcinoma cells, and provide solid evidence for the regulation of miR-223 by sulfatide.

我们预实验知道脑硫脂能促进整合素αV亚基的转录表达，而miR-223则对整合素αV亚基的表达有抑制作用，但是脑硫脂与整合素αV亚基之间的关系不清楚。本项目首先分析肝癌中miR-223与整合素αV的表达及其相关性；进而分析脑硫脂与miR-223表达的相关性，通过CST（脑硫脂合成关键酶）过表达和外源性脑硫脂的作用，检测和验证脑硫脂对miR-223的调控作用；为阐明作用机制，进一步检测脑硫脂对 miR-223基因启动子区域的乙酰化组蛋白H3和转录因子C/EBPα结合的影响，观察脑硫脂对H3k9/14和C/EBPα的乙酰化水平影响；检测脑硫脂对结合在miR-223启动子区乙酰基修饰酶（HBO1、MOZ和HDAC1）结合的影响。还构建miR-223和CST高表达的肝癌细胞株，进行裸鼠体内转移试验，验证对转移潜能的影响，并检测转移灶组织中miR-223、整合素αV并分析与脑硫脂的关系。

通过3年的研究工作，完成了本项目的研究内容，在肝癌病人的标本中发现了miR-223与整合素αV亚基的表达具有显著的相关性，在不同的细胞株中也存在着这种相关性; 在另一组103例肝癌病人的组织中，我们还发现脑硫脂的水平与miR-223的表达水平密切相关，进一步研究我们知道miR-223过表达能够显著抑制肝癌细胞中整合蛋白αV亚基和αVβ3的表达，并显著抑制肝癌细胞的迁移能力，在小鼠体内,miR-223过表达显著抑制肝癌的转移能力。 同时我们发现在肝癌细胞中异常表达的脑硫脂能够抑制miR-223的转录,从而促使合蛋白αV亚基的表达, 促进肝癌的转移。项目还探讨了脑硫脂对miR-223基因调控机制是通过减少启动子复合物中MOZ的占有，使转录活性降低。证明了脑硫脂是通过miR-223调控整合素αV的表达机制。通过本项目的研究阐明了miR-223的调控机理以及在肝癌转移过程中的作用，项目发表了5篇SCI论文，参加了6次全国和上海的学术会议，培养了3名研究生。