Abstract
BACKGROUND: Protein kinase D 3 (PRKD3), a serine/threonine protein kinase, functions as a crucial regulator across numerous cancer types. However, its regulatory function and mechanism in hepatocellular carcinoma (HCC) proliferation remain unclear. In vitro experiments and proteomics analysis offer new insights into the regulation and mechanism of PRKD3 in HCC. METHODS: A PRKD3 knockdown cell line was constructed to assess the effects of PRKD3 on proliferation of HCC cells using cell counting kit-8 (CCK-8) assay, 5-Ethynyl-2'-deoxyuridine (EdU) assay, clonogenic assay, and flow cytometry. Proteomic changes in liver cancer cells before and after PRKD3 knockdown were analyzed using 4D-lablefree technology. RESULTS: Analysis of The Cancer Genome Atlas (TCGA) dataset revealed abnormal PRKD3 expression in HCC, associated with poorer prognosis and specific pathological types. Results from the CCK-8 assay showed a marked reduction in the proliferation of Huh7 cells (P < 0.01), with the number of clonal colonies being 5.26 times higher than that in PRKD3 knockdown cells, and the EdU positivity rate decreased from 54.77% to 37.97%. Flow cytometry results indicated that PRKD3 knockout induced cell cycle arrest at G2/M phase. Proteomic analysis revealed 330 proteins had altered expression, associated with amino acid transport, stress response, and apoptosis. Cyclin-dependent kinase 4 (CDK4), plasminogen activator inhibitor 1 (SERPINE1), sequestosome 1 (SQSTM1), ras-related protein Rab-8A (RAB8A), and nuclear receptor-binding factor 2 (NRBF2) emerged as key nodes in the protein interaction network. CONCLUSION: This study elucidates the inhibitory effect of PRKD3 knockdown on HCC proliferation and unveils the proteomic features of PRKD3 regulation. CDK4, SERPINE1, SQSTM1, RAB8A, and NRBF2 may serve as key proteins in PRKD3's regulatory pathways.