PEP-1-mediated HNF4α Transduction And Its Effect On Hepatocellular Carcinoma Cells

Hepatocyte nuclear factor 4a (HNF4a) is a member of the nuclear hormone receptor superfamily. It plays an important role in the hepatocyte differentiation and hepatocellular carcinogenesis. Cell Penetrating Peptides (CPPs), consisted of 30 or less amino acids, are able to deliver cargoes across cellular membranes, such as proteins, RNA and DNA.In this study, we transduced recombinant hepatocyte nuclear factor 4 alpha (HNF4a) protein into hepatocellular carcinoma (HCC) cells mediated by cell penetrating peptide, PEP-1, and investigated the effect of the fusion protein P-HNF4a on HCC cells. Then the expression vector pET28a-P-HNF4a was constructed. The expression and purification conditions of fusion protein P-HNF4a were optimized. And P-HNF4a was acquired with concentration of 2.1μM and purity of 81% after concentrated by PEG and dialyzed with PBS. P-HNF4a was successfully transduced into HCC cells. The transduction efficiency of P-HNF4a has been calculated as 69.5% by Western blot, suggesting a high transduction efficiency of PEP-1. The sub-cellular localization of P-HNF4α was detected by Western blot with nuclear and cytoplasmic extracts from Huh7 cells transduced with P-HNF4aconfirmed by immunofluorescence assay. The result showed that P-HNF4a was located in the nucleus. Luciferase reporter assay indicated that P-HNF4a activated transcription via binding to its response element. Real-time RT-PCR showed that P-HNF4a up-regulated the expression of characteristic hepatocyte markers and down-regulated the "stemness" genes in Huh7 cells. Meanwhile, the cell proliferation, detected with CCK-8 kit, was dramatically inhibited by P-HNF4a. Moreover, the migration and invasion of HCC cells were significantly suppressed by P-HNF4a, which were detected by wound-healing assay and trans-well invasion assay, respectively.In conclusion, P-HNF4a induces the differentiation of HCC cells to mature hepatocytes, and inhibits the malignancy phenotype of HCC cells. Our result suggests that PEP-1-mediated HNF4a protein transduction may be a potential strategy of HCC differentiation therapy.