SOX7抑制肝癌发生的机制研究

We have reported that SOX7 could suppress hepatocarcinogenesis. However, its mechanism remains unclear. We found that SOX7 could inhibit β-catenin mRNA expression and promoter activity, and there were some putative SOX7 binding sites in the β-catenin promoter. On the other side, SOX7 could bind β-catenin to inhibit the interaction between β-catenin and TCF. In this study, we would explore the experiments such as 5´ sequential deletion analysis, EMSA, CHIP and CoIP, to obtain the evidence that SOX7 binding the β-catenin promoter to inhibit its transcription and SOX7 binding β-catenin to inhibit its interaction with TCF. We would also clarify that SOX7 suppresses hepatoma cell growth and hepatocarcinogenesis through negative regulation of β-catenin, and would analyze the correlation between SOX7, β-catenin and hepatocarcinogenesis. Finally, this study would declare a new mechanism that SOX7 negatively regulates β-catenin expression and activity by inhibiting transcription and by competitively inhibiting the interaction between β-catenin and TCF respectively, to suppress hepatocarcinogenesis. It could provide more scientific evidences for establishing SOX7 as a new therapeutic target for hepatocellular carcinoma.

申请人已报道SOX7能抑制肝癌发生，但其机制仍不清楚。预实验发现SOX7可以抑制β-catenin的mRNA表达和启动子活性，β-catenin启动子上有多个潜在SOX7结合位点；此外SOX7还可以结合β-catenin并抑制其与TCF结合。本项目拟采用5’末端序列续减分析、EMSA、CHIP、CoIP等技术，获得SOX7结合β-catenin启动子并抑制其转录表达和SOX7竞争性抑制β-catenin与TCF结合的证据，阐明SOX7通过负调控β-catenin抑制肝癌细胞生长及肝癌发生，分析肝癌组织中SOX7、β-catenin与肝癌发生的相关性，最终揭示“SOX7通过直接结合β-catenin启动子抑制其转录表达以及竞争性抑制β-catenin与TCF结合这两种机制，负调控β-catenin表达及活性，从而抑制肝癌发生”这一新机制，为确立SOX7为肝癌防治新靶点提供更充分的科学依据。

SOX7对肝癌发生具有重要的抑制作用，但其具体机制尚不清楚。本研究我们通过荧光定量PCR和双荧光素酶报道基因检测，发现SOX7可以抑制β-catenin的mRNA表达及启动子活性，进而利用5'末端序列续减分析我们确立了β-catenin启动子的核心区在转录起始点-196~-38之间，并且我们用生物信息学预测到核心区内的TTCAAGC位点为SOX7潜在结合位点，突变该位点后可以去除β-catenin启动子活性以及SOX7对β-catenin启动子活性的抑制作用，同时我们通过凝胶迁移实验和染色质免疫共沉淀实验在细胞外和细胞内证实了SOX7可以与该位点结合，从而证实了SOX7与β-catenin启动子的TTCAAGC序列结合并抑制β-catenin的转录；另一方面，我们还发现SOX7能结合β-catenin，并干扰β-catenin/TCF复合物形成及抑制β-catenin的活性，而突变与β-catenin的结合位点的SOX7Δ不能与β-catenin结合，也不会干扰β-catenin/TCF复合物形成及抑制β-catenin的活性，说明SOX7可以通过结合β-catenin干扰β-catenin/TCF复合物形成并抑制β-catenin的活性；我们还阐明了β-catenin可以拮抗SOX7对肝癌细胞生长及皮下肿瘤形成的抑制作用，而沉默β-catenin可以拮抗SOX7沉默对肝癌细胞生长及皮下肿瘤形成的促进作用，说明SOX7通过β-catenin发挥抗肿瘤生长作用；在肿瘤组织样本中，SOX7与β-catenin的蛋白表达呈负相关，SOX7表达与肿瘤大小、转移、血清AFP水平相关，β-catenin表达与肿瘤大小、分化、转移、血清AFP水平相关。综合上述实验结果，我们阐明了“SOX7通过直接结合β-catenin启动子抑制其转录表达以及竞争性抑制β-catenin与TCF结合这两种机制，负调控β-catenin表达及活性，从而抑制肝癌发生”这一新机制，为确立SOX7为肝癌防治新靶点提供更充分的科学依据。