转录中介因子1γ(TIF1γ)对肝癌干细胞异质性的调控作用与机制研究

Transcriptional intermediary factor 1γ (TIF1γ) is an important transcriptional regulator in the nucleus of mammalian cells. Our previous study revealed that reduced expression of TIF1γ was found in hepatocellular carcinoma (HCC) tissues when comparing with their adjacent noncancerous tissues. In addition, we found that TIF1γ played a dual role in HCC: It promoted tumor growth in early-stage HCC, but not in advanced-stage HCC, whereas it inhibited invasion and metastasis in both early- and advanced-stage HCC. We further found that TIF1γ inhibited the transcriptional activity of transforming growth factor-β / Drosophila mothers against decapentaplegic protein (TGF-β/ Smad) signaling in HCC, and the downstream effects of TGF-β /Smad signaling, such as regulating the proliferation, invasion and metastasis of HCC cells, were subsequently inhibited by TIF1γ. However, whether TIF1γ regulates signaling cascades and their downstream effects besides TGF-β/ Smad signaling in HCC is still needs further elucidation. We further analyzed our previous data and revealed that the downregulation of TIF1γ was more frequently found in poorly differentiated HCC, and we carried out spheroid assays of HCC cells and found that the expression of TIF1γ in HCC spheres (which enriches liver cancer stem cells, LCSCs) were decreased. In addition, we found that self-renewal and chemoresistance of HCC cells, both of which were important characteristics of LCSCs, were inhibited by TIF1γ. We further carried out flow cytometric analyses, Real-time PCR, Western blot analyses and results showed that TIF1γ enhanced the number of CD44+, CD24+ cells, whereas TIF1γ reduced EpCAM+ cell population in HCC. In addition, we found that TIF1γ regulated the expression of LCSC-related transcriptional factors. Specifically, TIF1γ upregulated the expression of KLF4 and c-myc, but downregulated the expression of Oct4 and SOX2. Previous studies demonstrated the existence of diverse LCSC populations with different markers in HCC, and the role of different LCSC populations in the progression of HCC was distinct. Previous investigations further proved the heterogeneity of liver cancer stem cells, that in different HCC patients, the distribution of LCSC populations was different, and this distribution determined the biological behavior and heterogeneity of HCC. From these results and our previous investigations we suppose that the expression of TIF1γ determines the distribution of LCSC populations, which means that TIF1γ regulates the heterogeneity of LCSCs. To confirm this hypothesis, we plan to carry out in vitro and in vivo experiments to investigate the regulatory role of TIF1γ in the heterogeneity of liver cancer stem cells and the related role of LCSCs in HCC. The results of purposed experiments will elucidate the precise regulatory role of TIF1γ in LCSCs and HCC, and could help us finding a novel strategy for HCC prevention and therapy targeting LCSCs.

转录中介因子1γ(TIF1γ)是一个重要的转录调控核因子。在肝癌中，我们的前期研究证实TIF1γ在癌组织中表达下降，这一趋势在低分化肝癌中尤为明显。此外,TIF1γ在肝癌细胞中调控TGFβ通路的转录活性并促进其增殖，却抑制其侵袭与转移。我们进而发现,TIF1γ在肝癌干细胞微球中表达降低，且抑制肝癌细胞的自我更新和对化疗药物耐药等干细胞特性。研究表明肝癌中存在多种具有不同标记物和生物学特性的肝癌干细胞群，这一特点构成了肝癌干细胞异质性。我们发现TIF1γ在肝癌细胞中上调EpCAM+，却下调CD44+，CD24+肝癌干细胞的数量，提高干细胞相关转录因子KLF4,c-myc，却降低Oct4,SOX2的表达。由此我们提出假说：肝癌中TIF1γ的表达水平决定肝癌干细胞群的构成进而决定了肝癌的生物学行为差异，即TIF1γ调控肝癌干细胞异质性。我们将通过体内外研究方案明确上述假说和TIF1γ的调控机制。

肝癌干细胞及肝癌细胞的“干性”是肝癌治疗后复发，对现有治疗耐药的重要原因。深入探索肝癌干细胞和肝癌的干性维持的分子机制可为开发针对肝癌干细胞群的靶向治疗药物及肝癌的诱导分化治疗提供理论基础。课题组前期对TIF1γ在肝癌中的表达模式和在肝癌转移中的抑制作用已进行相关研究，并以此为基础在本项目中继续深入研究TIF1γ在肝癌干细胞特征维持中的作用及分子机制。在研究中，课题组发现了TIF1γ可能调控肝癌免疫逃逸，并就这一新发现进行探索和拓展。关键发现如下：（1）TIF1γ抑制肝癌发生，肝癌干细胞微球形成和对化疗药物耐药等多种干细胞特性，且在具有干细胞特征的肝癌细胞中，TIF1γ的表达下降；（2）在肝细胞癌中TIF1γ可抑制干细胞相关转录因子SOX2和OCT4的表达，机制研究表明通过结合FACT蛋白复合物并抑制其对干细胞相关转录因子OCT4和SOX2的转录延长；（3）OCT4和SOX2通过直接结合TIF1γ启动子区域抑制TIF1γ表达；（4）TIF1γ抑制肝癌细胞中PD-L1表达并抑制PD-L1介导的免疫逃逸。上述结果对于理解肝癌干细胞特性维持的分子机制提供了重要依据。