Cell-permeated peptide P-T3H2 inhibits malignancy on hepatocarcinoma through stabilizing HNF4α protein. Cell-permeated peptide P-T3H2 inhibits malignancy on hepatocarcinoma through stabilizing HNF4α protein

Objectives: Hepatocyte nuclear factor 4α (HNF4α) is a key regulator of hepatocyte function and has a strong therapeutic effect on hepatocellular carcinoma (HCC) by inducing the differentiation of hepatoma cell into hepatocytes. Our previous study showed that Tribbles homolog 3 (TRIB3) directly interacts with and promotes the degradation of HNF4α in non-alcoholic fatty liver disease (NAFLD). Disrupting the TRIB3-HNF4α interaction by using a cell-permeating peptide called P-T3H2 stabilizes HNF4α protein. This study aimed to assess the anti-tumor impact of P-T3H2 in HCC. Methods: The expression of TRIB3 and HNF4α was assessed using western blot and immunohistochemistry staining (IHC). Hepatic functions in HCC cells were evaluated through periodic acid-Schiff (PAS) staining and acetylated low-density lipoprotein (ac-LDL) uptake. Senescence-associated β-galactosidase (SA-β-gal) activity staining was used to detected the cellular senescence in HCC cells. RNA-Seq analysis was carried out to identify differentially expressed genes in Huh-7 cells treated with the peptide P-T3H2 compared to the control peptide P-T3H2-2A. The impact of P-T3H2 on hepatocarcinoma malignancy was evaluated in HCC cells, a Huh-7 xenograft mouse model, an orthotopic model, and a C-MET/ΔN90-β-catenin-driven HCC model. Results: TRIB3 exhibited a negative correlation with HNF4α in both human and mouse HCC tissues. The administration of P-T3H2 significantly inhibited the malignancy of HCC cells. Additionally, P-T3H2 stabilized the HNF4α protein and facilitated the restoration of hepatic functions and the cellular senescence in HCC cells. RNA-Seq analysis demonstrated that P-T3H2 enhanced the transcriptional activity of HNF4α in HCC. Furthermore, P-T3H2 effectively suppressed the growth of HCC tumors in both of the Huh7 xenograft and orthotopic mouse models in vivo. Notably, P-T3H2 also inhibits hepatocarcinogenesis in the C-MET/ΔN90-β-catenin-driven HCC model. Overall design: To investigate the inhibitory effect of P-T3H2 on HCC by enhancing the transcriptional activity of HNF4α, Huh7 cells were treated with P-T3H2-2A and P-T3H2. We then performed gene expression profiling analysis using data obtained from RNA-seq of three groups of Huh7 cells were treated with P-T3H2-2A and P-T3H2.

研究目的：肝细胞核因子4α（Hepatocyte nuclear factor 4α, HNF4α）是肝细胞功能的关键调控因子，可通过诱导肝癌细胞向肝细胞分化，对肝细胞癌（hepatocellular carcinoma, HCC）发挥显著治疗作用。本团队前期研究发现，在非酒精性脂肪性肝病（non-alcoholic fatty liver disease, NAFLD）中，拟亲属同源物3（Tribbles homolog 3, TRIB3）可直接结合并促进HNF4α的降解。利用名为P-T3H2的细胞穿透肽破坏TRIB3与HNF4α的相互作用，能够稳定HNF4α蛋白。本研究旨在评估P-T3H2对肝细胞癌的抗肿瘤效果。
实验方法：采用蛋白质印迹（western blot）与免疫组织化学染色（immunohistochemistry staining, IHC）检测TRIB3与HNF4α的表达水平。通过过碘酸-希夫染色（periodic acid-Schiff, PAS）与乙酰化低密度脂蛋白（acetylated low-density lipoprotein, ac-LDL）摄取实验，评估肝癌细胞的肝细胞功能。采用衰老相关β-半乳糖苷酶（senescence-associated β-galactosidase, SA-β-gal）活性染色检测肝癌细胞的细胞衰老情况。对经P-T3H2肽与对照肽P-T3H2-2A处理的Huh-7细胞开展RNA测序（RNA-Seq）分析，以筛选差异表达基因。分别在肝癌细胞、Huh-7异种移植小鼠模型、原位肝癌模型以及C-MET/ΔN90-β-连环蛋白驱动的肝癌模型中，评估P-T3H2对肝癌恶性表型的影响。
实验结果：在人与小鼠的肝癌组织中，TRIB3的表达均与HNF4α呈负相关。给予P-T3H2可显著抑制肝癌细胞的恶性表型。此外，P-T3H2能够稳定HNF4α蛋白，促进肝癌细胞肝细胞功能的恢复与细胞衰老的发生。RNA测序分析显示，P-T3H2可增强HNF4α在肝癌细胞中的转录活性。进一步实验证实，P-T3H2可在体内有效抑制Huh-7异种移植与原位肝癌小鼠模型中的肿瘤生长。值得注意的是，P-T3H2同样可抑制C-MET/ΔN90-β-连环蛋白驱动的肝癌模型中的肝癌发生。
实验设计：为探究P-T3H2通过增强HNF4α转录活性以发挥抗肝癌作用的效应，我们将Huh-7细胞分别用P-T3H2-2A与P-T3H2进行处理。随后基于三组经P-T3H2-2A与P-T3H2处理的Huh-7细胞的RNA测序数据，开展基因表达谱分析。