Abstract: Objective To analyze the expression of TMEM64 in hepatocellular carcinoma (HCC) and investigate the effect of TMEM64 expression level on proliferation and invasion of HCC cells in vitro. Methods We analyzed the expression level of TMEM64 in HCC and adjacent tissues based on data from TCGA and GTEx databases. The prognostic value of TMEM64 for HCC patients was examined using Kaplan-Meier survival analysis and a Cox regression model, and a nomogram was constructed based on TMEM64 expression and clinical characteristics of the patients. Functional enrichment analysis was performed to explore the potential signaling pathways, and immune cell infiltration was assessed using single sample gene set enrichment analysis. We also performed cell experiment to observe the changes in proliferation, migration, and invasion in HCCLM3 cells with TMEM64 knockdown and in Huh7 cells with TMEM64 overexpression using CCK-8, EdU, colony formation, Transwell, and wound healing assays. Results The expression level of TMEM64 was significantly higher in HCC than in the adjacent tissues (P<0.05). Kaplan-Meier analysis suggested that a high expression of TMEM64 was associated with poor outcomes of the patients (P<0.05). Multivariate Cox regression analysis indicated that a high TMEM64 expression was an independent risk factor for overall survival of HCC patients (P<0.05). TMEM64 expression level was negatively correlated with the levels of immune cell infiltration by NK cells, CD8 + T cells, and plasma pDCs cells (P<0.05). GO, KEGG, and GSEA enrichment analyses showed that TMEM64 was significantly enriched with tumor invasion and metastasis pathways. The nomogram and calibration curves indicated a moderate prediction reliability of the model. In the cell experiment, TMEM64 knockdown obviously suppressed and TMEM64 overexpression markedly promoted the proliferation, migration, and invasion of HCC cells (P<0.01). Conclusion A high TMEM64 expression may serve as an independent risk factor for poor prognosis of HCC and promotes proliferation, migration, and invasion of HCC cells in vitro.

Key words: TMEM64; hepatocellular carcinoma; prognosis; invasion