The POR rs10954732 polymorphism decreases susceptibility to hepatocellular carcinoma and hepsin as a prognostic biomarker correlated with immune infiltration based on proteomics

Abstract

The effect of the cytochrome P450 oxidoreductase (POR) rs10954732 (G > A) polymorphism on hepatocellular carcinoma (HCC) susceptibility is unknown. Here we found that A allele carriers showed a 69% decrease in susceptibility to HCC with overall survival (OS) prolonged to 199%, accompanied by lower activity for cytochrome P450 2E1. A total of 222 differentially expressed proteins were mainly enriched in neutrophil and T cell activation and involved in the immune and inflammatory responses, constituting the altered immune tumor microenvironment related with A allele by proteomics analysis. Hepsin (HPN) showed significant down-regulation in HCC and up-regulation in A allele carriers. A lower HPN level was associated with increased susceptibility to HCC and a worse prognosis. Moreover, HPN is a potential independent prognostic biomarker for HCC and is strongly associated with clinicopathological features, tumor-infiltrating status of immune cells both in our discovery cohort and database surveys. Our findings provide a new potential mechanism by which HPN may play an important role in the susceptibility of rs10954732 A allele carriers to HCC and their prognosis through tumor immune infiltration, thus offering potential insights for future studies on tumor immunotherapy.

Keywords: HPN; Hepatocellular carcinoma; POR; Polymorphisms; Proteomics.

Fig. 1

An overview of the experimental design workflow. Illustration of the potential mechanism underlying the role of POR rs10954732 (G > A) polymorphisms in decreased HCC susceptibility and increased OS of HCC using label-free quantitative proteomic analysis. POR cytochrome P450 oxidoreductase, HCC hepatocellular carcinoma, OS overall survival time. A carriers refers to rs10954732 GA+AA genotypes; GG refers to wild type. DEPs differentially expressed proteins, HPN hepsin, AUC area under the ROC curves. Go enrichment for BP, assigning Gene Ontology (GO) terms to proteins, specifying involvement in biological processes (BP). Nor means normal group based on human liver samples. Con means control group based on HCC mouse model. RNA-seq RNA sequencing, HCC hepatocellular carcinoma, ELISA enzyme-linked immunosorbent assay, WB western blot, IHC immunohistochemistry

Fig. 2

POR rs10954732 polymorphisms related to decreased HCC susceptibility and increased OS of HCC patients as well as enzyme activity for CYP2E1 in healthy subjects and HCC patients. a Univariate logistic regression analysis of the association between rs10954732 polymorphisms and the risk of HCC. b Kaplan–Meier curve for overall survival (OS) of HCC patients between the GG genotype and GA + AA genotypes in HCC patients. Biochemical data (c) and CYP2E1 activity (d) in subjects between GG genotype and A allele carriers in healthy subjects and HCC patients. A allele carriers, GA + AA genotypes. Normal, healthy subjects. HCC, hepatocellular carcinoma. AST transaminase, ALT alanine aminotransferase, V_max maximum reaction velocity, CL_int intrinsic clearance. Data are expressed as mean ± SEM, P value was calculated by Mann–Whitney U test

Fig. 3

Proteomic mechanisms for POR rs10954732 polymorphisms to confer decreased susceptibility to HCC. a A total of 222 DEPs related to rs10954732 A allele compared with the GG genotype in HCC patients as shown in a heat map. GO enrichment analysis (b) and Volcano plot (c) for 222 DEPs with dysregulation related with A allele carriers. d Wayne figure shows 46 DEPs intersections between 222 A allele-related DEPs and 1342 HCC-related DEPs. e Heat map of 34 DEPs with protein changes showing the opposite direction in the HCC group and A allele carriers. f Expression profiles of the top eight significantly up-regulated and one down-regulated DEPs in peritumoral tissues of HCC. Data are shown as mean ± SEM, **P < 0.01, ***P < 0.001 vs Normal group by Mann–Whitney U test. DEPs differentially expressed proteins, HCC hepatocellular carcinoma

Fig. 4

Significantly decreased HPN expression in HCC and associated with worse prognosis. a Decreased protein abundance of HPN in HCC patients and an ROC curve of HPN for diagnosis of HCC in the discovery cohort. b Increased protein abundance of HPN with rs10954732 GA + AA genotypes in HCC patients. c Low expression of HPN conferring a poor prognosis in overall HCC patients in the discovery cohort. d Significantly altered OS between HPN high-expressing and low-expressing groups only in A allele carriers. e Patients with higher HPN levels were more likely to have lower AST, ALT, GGT and GLO. f Differential expression and ROC curve of HPN in adjacent tissues in HCC patients in TCGA database by comparison with normal subjects in the GTEx database. g Levels of HPN expression in tumor tissues of different tumor types from the TCGA database in TIMER. Note: *P < 0.05, **P < 0.01, ***P < 0.001. HCC hepatocellular carcinoma, AST transaminase, ALT alanine aminotransferase, GGT γ-glutamyl transpeptadase, GLO globulin

Fig. 5

Prognostic value of highly-expressed and lowly-expressed HPN in HCC patients. a, b Low HPN expression was correlated with worse OS in the TCGA database (a) and the GEPIA database (b). Survival curves of OS (c) and PFS (d) in HCC using the Kaplan–Meier plot database (n = 364, n = 370). Correlation of HPN mRNA expression and OS (e) and PFS (f) in HCC with different clinicopathological factors by Kaplan–Meier plot (n = 364, n = 370). OS overall survival, PFS progression-free survival

Fig. 6

Correlation analysis of HPN expression and infiltration levels of immune cells in HCC tissues. a Correlation analysis of HPN abundance and expression of markers of immune cells based on proteomic analysis in the discovery cohort (n = 60). b Correlation between HPN expression and infiltration levels of immune cells in HCC by the TIMER database (n = 371). c Correlation between HPN level and twenty-eight tumor immune infiltrating cell subtypes among human heterogeneous cancers. d Top four immune cell subtypes with the greatest Spearman’s correlation value for HPN expression in HCC. HCC hepatocellular carcinoma

Fig. 7

Kaplan–Meier survival curves between HPN high- and low-expressing groups in HCC based on immune cell subgroups. Low HPN level in enriched B cells (a), CD4 + memory T cells (b), CD8 + T cells (c), NK T cells (e), Treg cells (f), Th1 cells (g) and Th2 cells (h) is correlated with worse OS in HCC. For the enriched macrophages, low HPN level showed a decreasing trend in OS, though it did not reach statistical significance. HCC hepatocellular carcinoma

Fig. 8

Verification of decreased HPN expression in HCC and correlation with expression of marker genes for immune cells. a Western blot analysis of HPN in another cohort of human samples. General picture (b), representative images of H&E staining (c) and western blot analysis of HPN (d) in a BALB/c mouse model. e Representative images of immunohistochemical staining for CD68, CD163 and IL-10 in a BALB/c mouse model (Scale bar, 100 μm). Data are expressed as mean ± SEM. Statistical analysis was performed according to a Mann–Whitney test. Normal means healthy subjects based on human liver samples. Control means control group based on the HCC mouse model. H&E, haematoxylin and eosin. **P < 0.01, ***P < 0.001 vs Normal or Control. HCC hepatocellular carcinoma. (n = 6 for each group)