关键词: 三白草酮, 肝癌, 免疫逃逸, Krüppel样因子5-促红细胞生成素肝细胞A2通路

Abstract:

Objective To investigate the effects of sauchinone （Sch） on the growth and immune escape of liver cancer cells by regulating the Krüppel like factor 5 （KLF5）-erythropoietin-producing hepatocellular receptor A2 （EphA2） pathway. Methods Huh-7 cells were grouped into liver cancer group， Sch low， medium， and high-dose groups （Sch-L group， Sch-M group， Sch-H group）， Sch-H + KLF5 activator negative control group （OE-NC）， and Sch-H + KLF5 activator （OE-KLF5） group. 5-bromo-2-deoxyuracil （EdU） staining， CCK-8， scratch assay， and Transwll were used to detect the proliferation， migration， and invasion of Huh-7 cells， respectively. Western blot was applied to detect proliferative cell nuclear antigen （PCNA）， migration invasion enhancer（MIEN1）， matrix metalloproteinase-2 （MMP-2）， programmed death receptor ligand 1 （PD-L1）， KLF5， and EphA2 proteins in Huh-7 cells. The Huh-7 cells in above 6 groups were co cultured with activated CD8⁺ T cells in a 96 well plate and named as liver cancer co culture group， Sch-L co culture group， Sch-M co culture group， Sch-H co culture group， Sch-H + OE-NC co culture group， and Sch-H + OE-KLF5 co culture group. The killing rate of CD8⁺T cells in the co culture system and the levels of interferon-γ （IFN-γ）， interleukin-4 （IL-4）， and tumor necrosis factor-α （TNF-α） in the supernatant were detected. Results Compared with the liver cancer group， the EdU positive rate， OD₄₅₀ value， cell invasion number， and PCNA， MIEN1， MMP-2， PD-L1， KLF5， and EphA2 proteins in the Sch-L， Sch-M， and Sch-H groups reduced， the migration distance shorten （P < 0.05）. Compared with the Sch-H group and Sch-H + OE-NC group， the EdU positive rate， OD₄₅₀ value， cell invasion number， and PCNA， MIEN1， MMP-2， PD-L1， KLF5， and EphA2 proteins in the Sch-H + OE-KLF5 group increased， the migration distance prolonged （P < 0.05）. Compared with the liver cancer co culture group， the killing rate of CD8⁺T cells on Huh-7 cells and the levels of IFN-γ， TNF-α， and IL-4 in the supernatant in the Sch-L co culture group， Sch-M co culture group， and Sch-H co culture group increased （P < 0.05）. Compared with the Sch-H co culture group and the Sch-H + OE-NC co culture group， the killing rate of CD8⁺ T cells on Huh-7 cells and the levels of IFN-γ， TNF-α， and IL-4 in the supernatant in the Sch-H + OE-KLF5 co culture group decreased （P < 0.05）. Conclusion Sch may inhibit growth and immune escape of liver cancer cells by inhibiting the KLF5-EphA2 pathway.

Key words: sauchinone, liver cancer, immune escape, krüppel like factor 5-erythropoietin-producing hepatocellular receptor A2 pathway