To select suitable human HCC cell lines for subsequent functional investigations, the mRNA expression and secretion levels of SPINK1 were assessed across various HCC cell lines. The analysis led to the selection of the SPINK1high Huh7 cell line for repression and the SPINK1low MHCC97L cell line for overexpression of SPINK1. Subsequently, the validation of SPINK1 expression was performed by performing western blot analysis on Huh7 cells with SPINK1 knockdown and MHCC97L cells treated with recombinant SPINK1.