〔Abstract〕 Objective To investigate the regulatory role of ubiquitin-specific protease 10(USP10) on the protein expression of Krüppel-like factor 4(KLF4) and its impact on the proliferation and invasion ability of hepatocellular carcinoma(HCC) cells. Methods The protein expression differences of USP10 and KLF4 in normal liver cell line L02 and HCC cell lines, including HepG2,HUH7,HCCLM3 were detected by immunoblotting(Western blot) methods.HCCLM3 and HUH7 cells were selected, and lentiviral particles overexpressing or silencing USP10(oe-USP10 or sh-USP10) was transfected into the cells, and they were designated as the oe-USP10 group and oe-NC group, respectively.Immunoprecipitation(Co-IP) experiments were conducted to examine whether USP10 could directly interact with KLF4 in HCCLM3 or HUH7 cells.The Co-IP assay was repeated in HCC cells transfected with oe-USP10 or sh-USP10,with the addition of the proteasome inhibitor MG132,which used to detect the ubiquitination level of KLF4 protein in the transfected HCC cells.The pcDNA3.1 vector containing overexpressed KLF4 or its negative control plasmid(pc-KLF4 or pc-NC) was co-transfected into cells of the sh-USP10 group or sh-NC group.These cells were designated as the sh-NC+pc-NC group, sh-USP10+pc-NC group, sh-NC+pc-KLF4 group, and sh-USP10+pc-KLF4 group.The cell proliferation activity of each group was measured using the CCK-8 assay, and the cell invasion ability was assessed using the Transwell assay. Results Compared to L02 cells, the protein expression of USP10 and KLF4 significantly decreased in HepG2,HUH7,HCCLM3,and other cells(P<0.05).In HCCLM3 and HUH7 cells, USP10 protein directly interacted with KLF4.Furthermore, treatment with MG132 resulted in a time-dependent increase in KLF4 protein expression in HCCLM3 and HUH7 cells.Silencing USP10 increased the ubiquitination of KLF4 in HCCLM3 or HUH7 cells, while overexpressing USP10 decreased the ubiquitination level of KLF4 in cells.Compared to the sh-NC+pc-NC group, both the proliferation activity and invasion ability of HCCLM3 and HUH7 cells significantly increased in the sh-USP10+pc-NC group(P<0.01),while they significantly decreased in the sh-NC+pc-KLF4 group and sh-USP10+pc-KLF4 group(P<0.05).Compared to the sh-USP10+pc-NC group, the proliferation activity and invasion ability of cells significantly decreased in the sh-USP10+pc-KLF4 group(P<0.05). Conclusion USP10 can promote the stability of KLF4 protein through deubiquitination in HCC cell lines, thereby inhibiting the proliferation and invasion of tumor cells.