Single-stranded DNA binding protein 1 (SSBP1) is a DNA binding protein found in mitochondria, encoded by nuclear genes. SSBP1 plays a crucial role in responding to mitochondrial DNA (mtDNA) damage and maintaining genome stability, and it is linked to cancer occurrence and progression, but its role in hepatocellular carcinoma (HCC) is still unclear. Therefore, the

High expression of SSBP1 is an independent risk factor for poor prognosis in HCC patients and has good diagnostic value.

RNA-seq data and clinical information of HCC samples and normal liver samples were downloaded from The Cancer Genome Atlas (TCGA). The expression of SSBP1 in HCC and its correlation with clinical pathological indicators, prognosis, immune cells, and infiltration were analyzed using R software, while the diagnostic value of SSBP1 in HCC was evaluated. Using the R software, we conducted Gene Ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and gene set enrichment analysis (GSEA) on the SSBP1 expression in HCC. Immunohistochemistry (IHC) detected SSBP1 expression in 31 HCC tissue pairs. Western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) quantified protein and mRNA levels in 6 fresh HCC tissue. Protein expression and distribution of SSBP1 in HCC cell lines were analyzed using qRT-PCR, Western blotting, and Immunofluorescence techniques.

SSBP1 mRNA expression was significantly higher in HCC tissues compared to normal tissues (P<0.001) in both matched and unmatched samples. SSBP1 expression was correlated with gender and M stage (P<0.05), but not with other factors. High SSBP1 expression was identified as an independent risk factor for overall survival (OS) in HCC patients [hazard ratio =1.713, P=0.01]. Immunocell infiltration analysis showed a negative correlation between SSBP1 expression and level of naive B cells, but a positive correlation with memory B cells and macrophages (|Spearman's r| >0.2, P<0.05). The diagnostic value of SSBP1 mRNA expression for early diagnosis prognosis of HCC (area under the curve >0.50). The GO enrichment analysis of SSBP1 revealed that it was enriched for mitochondrial biological functions. KEGG analysis showed that SSBP1 was associated with multiple DNA replication, mismatch repair and homologous recombination pathways. GSEA analysis showed that the first three pathways strongly related to the high expression of SSBP1 were DNA repair, myc-targets-v1 and reactive oxygen species signaling pathways. Validation through IHC and Western blotting high SSBP1 protein expression in HCC tissue, as well as qRT-PCR and Western blotting results showed high expression in HCC cell lines. Immunofluorescence experiments indicated the localization of SSBP1 in the mitochondria of HCC cells. Conclusions: High expression of SSBP1 is an independent risk factor for poor prognosis in HCC patients and has good diagnostic value.