USP7 Enhances Epithelial Mesenchymal Transition and Cancer Stem Cell-Like Properties in Hepatocellular Carcinoma by the AKT/β-Catenin Pathway: A Randomized Trial

Abstract

Background: The malignant progression of hepatocellular carcinoma (HCC) is closely linked to the epithelial-mesenchymal transition (EMT) process and cancer stem cells (CSCs) characteristics. Ubiquitin-specific protease 7 (USP7) is found to be elevated in various tumors, but its molecular mechanism for regulating EMT and CSCs in HCC has not been fully elucidated. Objectives: To investigate whether USP7 affects the EMT process and CSCs characteristics in HCC cells via regulation of the AKT/β-catenin pathway. Methods: Twenty-four Balb/c nude mice were randomly assigned to the OE-USP7, OE-NC, sh-USP7, or sh-NC groups using a random number table method (n=6 mice in each group), and 200 μL of Huh-7 cell suspension transfected with OE-USP7, OE-NC, sh-USP7, or sh-NC was injected into mice to establish the HCC model, respectively. The OE-NC group and sh-NC group served as controls for the OE-USP7 and sh-USP7 groups, respectively. Blinding procedures were implemented throughout the experiment: the researchers responsible for nude mouse feeding and tumor growth monitoring (measuring tumor volume weekly) were blinded to the grouping information. On the 28th day, mice were euthanized, and the proliferation marker Ki-67 in tumors was assessed by immunohistochemistry. Immunofluorescence and Western blot detected USP7 expression in THLE-2, HCCLM3, and Huh-7 cells. Cell proliferation, migration, invasion ability, and stemness were examined through cell counting kit-8 (CCK-8), Transwell, and sphere formation assays. Flow cytometry was utilized to analyze CSCs markers. Western blot detected AKT/β-catenin pathway proteins, EMT markers, and stemness factors levels. Results: Compared with normal hepatocytes, USP7 level in HCC cells was remarkably higher (P < 0.05). Overexpression of USP7 increased cell viability, globulation, and CSCs characteristics, and facilitated migratory invasion and EMT process in HCC cells, while knockdown of USP7 inhibited the above phenotypes (P < 0.05). Overexpression of USP7 activated the AKT/β-catenin pathway, but knockdown of USP7 blocked this pathway. PI3K inhibitors attenuated the enhancing impact of overexpression of USP7 on the EMT process and CSCs characterization in HCC cells (P < 0.05). Additionally, knockdown of USP7 reduced both the size and weight of tumor tissues in vivo, decreased the Ki67 positivity rate, and inhibited the EMT process and CSCs characteristics. Conclusions: USP7 enhances the properties of CSCs and promotes cell proliferation and EMT process in HCC cells through regulation of the AKT/β-catenin pathway.