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.

中文翻译：

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过度表达的 GNAZ 可预测肝细胞癌的不良预后并促进 G0/G1 细胞周期进展

宗旨

我们旨在研究 G 蛋白亚基 α Z(GNAZ) 在肝细胞癌 (HCC) 患者的进展和预后中的作用。

方法

使用Oncomine、GEO、TCGA、GEPIA2、Kaplan-Meier Plotter、TIMER2、Metascape、CCLE、LinkedOmics和UALCAN数据库分析GNAZ在HCC和正常肝组织中的差异表达、GNAZ表达与HCC患者预后的关系以及 GNAZ 在常见人类 HCC 细胞系中的表达。进行Western印迹分析GNAZ表达，同时使用Cell Counting Kit 8测定法测定细胞增殖，并使用流式细胞术评估细胞周期和凋亡。伤口愈合和 transwell 侵袭试验用于研究细胞转移和侵袭。

结果

使用 Oncomine、基因表达综合 (GEO) 和 GEPIA2 数据库，发现与邻近正常肝组织相比，GNAZ 在 HCC 组织中过表达，并且蛋白质印迹分析显示 GNAZ 在接受 HCC 手术切除的 7 名 HCC 患者中过表达和第-cancerous组织（p  <0.01）。生存分析显示，高 GNAZ 表达与 HCC 患者的总生存期（OS）、无复发生存期、无进展生存期和疾病特异性生存期呈负相关（p < 0.05)。GNAZ 过度表达与更差的 4 个月、6 个月、12 个月、24 个月、36 个月、48 个月和 60 个月的 OS，以及 HCC 患者的不同临床病理特征相关，包括肝炎病毒感染状态；饮酒状态；男性; 女性; 亚洲人；微血管侵犯、I-II 期、II-III 期和 III-IV 期；和 II 级（Cox 回归，p < 0.05)。KEGG/GO生物过程富集表明HCC中与GNAZ相似的基因主要富集在细胞周期、细胞周期相变、DNA复制检查点、G0到G1过渡的调控等方面。siRNA-GNAZ 显着降低了 JHH-2 和 SNU-761 细胞的活力，从 12 小时到 96 小时；增加G0/G1期细胞百分比，降低S期和G2/M期细胞百分比（p  < 0.05）；并显着下调细胞周期蛋白 D、细胞周期蛋白 E 和 CDK2 蛋白的表达。siRNA-GNAZ还显着增加了晚期JHH-2和SNU-761细胞凋亡的百分比，而存活细胞数量减少（p < 0.05)，并上调凋亡相关蛋白 Bax 和 caspase 3 蛋白的表达。此外，与对照组相比，siRNA-GNAZ显着降低了JHH-2和SNU-761细胞划痕伤口的愈合和侵袭细胞的数量（p  < 0.001）。

结论

我们的研究表明，GNAZ作为潜在的致癌基因发挥着关键作用，并可预测 HCC 患者的不良预后。它通过细胞周期停滞、细胞凋亡、迁移和侵袭促进肿瘤增殖。因此，GNAZ可能是一种潜在的候选生物标志物，可提供对肝癌发生和侵袭性的有用见解。