PEP-1介导的重组肝细胞核因子4α蛋白转导对肝癌细胞的抑制作用

Inhibitory effect of PEP-1-mediated recombinant hepatocyte nuclear factor 4 alpha transduction on hepatocellular carcinoma cells

目的利用细胞穿膜肽PEP-1介导重组肝细胞核因子4α(HNF4α)蛋白进入肝癌细胞,并明确外源融合蛋白PHNF4α对肝癌细胞的作用。方法构建表达质粒pET28a-P-HNF4α,优化原核表达体系的诱导条件,经大量表达、亲和层析纯化及浓缩、透析后获得纯度较高的带有细胞穿膜肽PEP-1的融合蛋白P-HNF4α;P-HNF4α转导人肝癌细胞,蛋白质印迹法检测其穿膜效率,核质分离和细胞免疫荧光检测P-HNF4α的亚细胞定位,Real-time PCR检测肝癌细胞基因表达,CCK-8法检测肝癌细胞增殖,细胞划痕实验及小室侵袭实验检测P-HNF4α对肝癌细胞转移能力的影响。结果细胞穿膜肽PEP-1成功介导融合蛋白P-HNF4α进入Huh7细胞并定位于细胞核;P-HNF4α蛋白可促进Huh7细胞肝功能基因表达,抑制干细胞相关基因表达(P<0.05或0.01),并显著抑制肝癌细胞增殖(P<0.05)、迁移(P<0.001)和侵袭(P<0.05)能力。结论 P-HNF4α可诱导肝癌细胞向成熟肝细胞分化,降低肝癌细胞的恶性程度,是诱导分化治疗肝癌的潜在手段。

Objective To investigate cell penetrating peptide （PEP-l） mediated transduction of recombinant hepatocyte nuclear factor 4 alpha （HNF4α） protein into hepatocellular carcinoma （HCC） cells, and to observe the effect of the fusion protein P-HNF4α on HCC cells. Methods The expression vector pET28-P-HNF4α was constructed. The prokaryotic expression condition of fusion protein P HNF4α was optimized. Recombinant P-HNF4α carrying cell penetrating peptide PEP-1 was obtained by abundant expression, purified by affinity chromatography, and was concentrated and dialyzed. P-HNF4α was transduced into HCC cells. The transduction efficiency was analyzed by Western blotting analysis. Sub-cellular localization of P-HNF4α was detected by Western blotting analysis with nuclear and cytoplasmic extracts and confirmed by immunofluorescence assay. Real-time RT-PCR was used to examine the gene expression of HCC cells. The proliferation of HCC cells was detected with CCK-8 kit. The migration and invasion of HCC cells were detected by wound-healing assay and trans- well invasion assay, respectively. Results P-HNF4α was efficiently transduced into Huh7 cells and located in the nucleus as mediated by PEP-1. P-HNF4α significantly up-regulated the expression of characteristic hepatocyte markers and down-regulated the ＂sternness＂ genes in Huh7 cells （P〈0. 05 or P〈0. 01）. Moreover, the proliferation （P〈0. 05）, migration （P〈0. 001） and invasion （P〈0. 05） of HCC cells were significantly suppressed by fusion protein P-HNF4α. Conclusion P-HNF4a can induce the differentiation of HCC cells to mature hepatocytes and reduce the malignancy phenotype of HCC cells, suggesting that PEP-l-mediated HNF4a protein transduction may be a potential strategy for HCC differentiation therapy.