Abstract:

Objective To discuss the regulatory effect of keratin 23 （KRT23） on the stem cell activity in hepatocellular carcinoma （HCC）， and to clarify its mechanism. Methods The Gene Expression Profiling Interactive Analysis （GEPIA） database was used to analyze the expression of KRT23 in hepatocellular carcinoma tissues and normal liver tissues； Kaplan-Meier Plotter survival analysis was performed to compare the survival of hepatocellular carcinoma patients with high and low expression of KRT23， and to evaluate the impact of KRT23 on the prognosis of hepatocellular carcinoma patients. Second-generation sequencing analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） signaling pathway analysis were conducted to identify the signaling pathways potentially regulated by KRT23， and the Wnt/β-catenin signaling pathway was finally selected for mechanistic study. Real-time fluorescence quantitative PCR （RT-qPCR）， Western blotting， and immunohistochemistry methods were used to verify the expression of KRT23 in HCC cells and HCC tissue， respectively， and immunofluorescence was used to clarify its subcellular localization. A lentiviral vector for KRT23 knockdown was constructed using a small interfering RNA （siRNA） with verified knockdown efficiency. After knocking down the expression level of KRT23， the stem cell sphere formation assay was performed to verify the regulatory effect of KRT23 on hepatocellular carcinoma stem cell activity， and RT-qPCR and Western blotting methods were used to detect the changes of related stem cell markers. Immunofluorescence and nuclear-cytoplasmic protein extraction were further used to verify the regulation effect of KRT23 on stem cells related indicators in the Wnt/β-catenin signaling pathway. Results The expression level of KRT23 in hepatocellular carcinoma tissues was higher than that in normal liver tissue. Compared with immortalized hepatocyte LO2， the expression levels of KRT23 in hepatocellular carcinoma cells were increased （P<0.05）； compared with the hepatocellular carcinoma patients with low expression of KR23， the hepatocellular carcinoma patients with high expression of KRT23 had a poorer prognosis （P=0.038）. The second-generation sequencing and KEGG signaling pathway analysis results showed that KRT23 might regulate stem cell activity and participate in the Wnt/β-catenin signaling pathway. The immunofluorescence localization results showed that KRT23 was mainly located in the cytoplasm. After verification of the knockdown efficiency of siRNA targeting KRT23， a lentiviral vector for KRT23 knockdown was constructed， and the transfection efficiency was re-verified. After KRT23 expression was reduced， compared with sh-NC group， the stem cell sphere formation rate in sh-KRT23 group was significantly decreased （P<0.01）， and the expression levels of stem cell-related markers cluster of differentiation 133 （CD133）， cluster-of-differentiation 44（CD44）， pctamer-blinding transcription factor 4（OCT4）， and SRY-box transcription-factor 2（SOX2） were also decreased （P<0.05 or P<0.01）. Conclusion KRT23 enhances the stem cell activity in hepatocellular carcinoma by regulating the Wnt/β-catenin signaling pathway， and the mechanism is related to the activation of the Wnt/β-catenin signaling pathway.

Key words: Keratin 23, Liver neoplasm, Stem cell activity, Cluster of differentiation 133, Pctamer-blinding tramscription factor 4