Overexpressed GNAZ predicts poor outcome and promotes G0/G1 cell cycle progression in hepatocellular carcinoma

Aims: We aimed to investigate the role of G protein subunit alpha Z(GNAZ) in the progression and prognosis of patients with hepatocellular carcinoma (HCC).

Methods: Oncomine, GEO, TCGA, GEPIA2, Kaplan-Meier Plotter, TIMER2, Metascape, CCLE, LinkedOmics, and UALCAN databases were used to analyze the differential expression of GNAZ in HCC and normal liver tissues, relationship between GNAZ expression and prognosis of patients with HCC, and expression of GNAZ in common human HCC cell lines. Western blotting was performed to analyze GNAZ expression, while the Cell Counting Kit 8 assay was used to determine cell proliferation, and flow cytometry was used to evaluate the cell cycle and apoptosis. Wound healing and transwell invasion assays were used to investigate cell metastasis and invasion.

Results: Using Oncomine, Gene Expression Omnibus (GEO), and GEPIA2 databases, GNAZ was found to be overexpressed in HCC tissues compared with that in adjacent normal liver tissues, and western blotting analysis showed GNAZ overexpression in seven patients with HCC who underwent surgical resection of HCC and para-cancerous tissues (p < 0.01). Survival analysis revealed that high GNAZ expression was negatively associated with overall survival (OS), recurrence-free survival, progression-free survival, and disease-specific survival in patients with HCC (p < 0.05). GNAZ overexpression was associated with worse 4- month, 6- month, 12- month, 24- month, 36- month, 48- month, and 60-month OS, as well as with different clinicopathological characteristics of patients with HCC, including hepatitis virus infection state; alcohol consumption state; male; female; Asian; microvascular invasion, Stage I-II, Stage II-III, and Stage III-IV; and grade II (Cox regression, p < 0.05). KEGG/GO biological process enrichment indicated that the genes similar to GNAZ in HCC were mainly enriched in the cell cycle, cell cycle phase transition, DNA replication checkpoint, and regulation of G0 to G1 transition. siRNA-GNAZ significantly reduced the viability of JHH-2 and SNU-761 cells from 12 to 96 h; increased the percentage of cells in the G0/G1 phase and decreased that of cells in the S and G2/M phases (p < 0.05); and markedly downregulated the expression of cyclin D, cyclin E, and CDK2 protein. siRNA-GNAZ also significantly increased the percentage of JHH-2 and SNU-761 cell apoptosis at late stages, while the number of surviving cells decreased (p < 0.05), and upregulated the expression of apoptosis-related proteins Bax and caspase 3 protein. Furthermore, siRNA-GNAZ remarkably reduced the healing of scratch wounds in JHH-2 and SNU-761 cells and the number of invasive cells compared with that in the control group (p < 0.001).

Conclusion: Our study demonstrated that GNAZ plays a pivotal role as a potential oncogene and predicts poor prognosis in patients with HCC. It promotes tumor proliferation via cell cycle arrest, apoptosis, migration, and invasion. Thus, GNAZ may be a potential candidate biomarker providing useful insight into hepatocarcinogenesis and aggressiveness.