Exploring the Prognostic Value, Immune Implication and Biological Function of H2AFY Gene in Hepatocellular Carcinoma

Abstract

Background: Hepatocellular carcinoma (HCC) is an extremely malignant cancer with poor survival. H2AFY gene encodes for a variant of H2A histone, and it has been found to be dysregulated in various tumors. However, the clinical value, biological functions and correlations with immune infiltration of H2AFY in HCC remain unclear.

Methods: We analyzed the expression and clinical significance of H2AFY in HCC using multiple databases, including Oncomine, HCCDB, TCGA, ICGC, and so on. The genetic alterations of H2AFY were analyzed by cBioPortal and COSMIC databases. Co-expression networks of H2AFY and its regulators were investigated by LinkedOmics. The correlations between H2AFY and tumor immune infiltration were explored using TIMER, TISIDB databases, and CIBERSORT method. Finally, H2AFY was knocked down with shRNA lentiviruses in HCC cell lines for functional assays in vitro.

Results: H2AFY expression was upregulated in the HCC tissues and cells. Kaplan-Meier and Cox regression analyses revealed that high H2AFY expression was an independent prognostic factor for poor survival in HCC patients. Functional network analysis indicated that H2AFY and its co-expressed genes regulates cell cycle, mitosis, spliceosome and chromatin assembly through pathways involving many cancer-related kinases and E2F family. Furthermore, we observed significant correlations between H2AFY expression and immune infiltration in HCC. H2AFY knockdown suppressed the cell proliferation and migration, promoted cycle arrest, and apoptosis of HCC cells in vitro.

Conclusion: Our study revealed that H2AFY is a potential biomarker for unfavorable prognosis and correlates with immune infiltration in HCC.

Keywords: H2AFY; biomarker; hepatocellular carcinoma; immune infiltration; prognosis.

Figure 1

The elevated H2AFY expression in HCC. (A) Upregulated or downregulated H2AFY expression in different cancer types (Oncomine database, red color—upregulation, blue color—downregulation). (B) H2AFY expression levels in different tumor tissues and normal tissues (TIMER database). (C) Comparing the H2AFY expression between HCC and adjacent tissues in ten HCC cohorts (HCCDB database) *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

H2AFY expression in sub-groups of different clinical characteristics. Subgroup analyses of H2AFY expression based on (A) age, (B) survive status, (C) gender, (D) histological grade, (E) tumor stage, and (F) T classification.

Figure 3

Genetic alterations of H2AFY in HCC. (A) OncoPrint of H2AFY alterations in TCGA-LIHC cohort (cBioPortal). (B) Schematic presentation of H2AFY mutations in TCGA-LIHC cohort (cBioPortal). (C, D) The mutation types of H2AFY in HCC (Catalogue of Somatic Mutations in Cancer (COSMIC) database).

Figure 4

H2AFY is associated with overall survival of HCC patients. (A) Kaplan–Meier survival curves and (B) time-dependent ROC curves of H2AFY in TCGA-LIHC cohort. (C) univariate Cox analysis and (D) multivariate Cox analysis in TCGA-LIHC cohort. (E) Kaplan–Meier survival curves and (F) time-dependent ROC curves of H2AFY in ICGC cohort (LIRI-JP project). (G) Kaplan–Meier survival analyses of H2AFY in Kaplan–Meier Plotter and (H) GEPIA2.

Figure 5

H2AFY co-expression networks in HCC (LinkedOmics). (A) Volcano plot of the global H2AFY highly correlated genes identified by Spearman test. (B) Heat maps of top 50 genes positively and negatively correlated with H2AFY. (C) Survival heatmaps of top 50 genes positively and negatively correlated with H2AFY. (D) Significantly enriched GO terms and KEGG pathways related to H2AFY.

Figure 6

GSEA in TCGA-LIHC cohort. (A, B) The GO_BP annotations enriched in HCC patients with high/low H2AFY expression. (C, D) The KEGG pathways enriched in HCC patients with high/low H2AFY expression.

Figure 7

Correlations of H2AFY expression with immune infiltration in HCC. (A) Correlation analysis of H2AFY expression and abundance of immune cells in TIMER. (B) H2AFY copy number alterations affects the immune infiltration levels. (C) Correlations between the expression of H2AFY and several immune checkpoint genes. (D) The expression of several immune checkpoint genes between high- and low-H2AFY expression patients. (E) H2AFY expression in different immune subtypes of HCC (TISIDB database) *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 8

Correlation of H2AFY expression and 22 immune cell types in HCC based on CIBERSORT. (A) The relative fraction of 22 immune cell types in TCGA-LIHC cohort. (B) The heat map showing relative immune cell fraction of HCC patients (C) Violin plots showing the difference of 22 immune cell types between high- and low-H2AFY expression patients *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 9

Effects of H2AFY knockdown on cell proliferation and apoptosis in HCC cells. (A) H2AFY mRNA expression in normal liver cell line (L02) and several HCC cell lines. (B) Evaluation of H2AFY expression in HepG2 and Hep3B cells after shRNA transfection. (C) The effect of H2AFY knockdown on cell proliferation in HepG2 and Hep3B cells examined by CCK8 assay and (D) colony formation assay. (E) The effect of H2AFY knockdown on cell apoptosis in HepG2 and Hep3B cells examined by flow cytometry ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 10

Effects of H2AFY knockdown on cell cycle and migration in HCC cells. (A) Cell cycle detected by flow cytometry in HepG2 and Hep3B cells after H2AFY knockdown. (B–D) Representative images of transwell (200×) and wound healing assays (40×) in HepG2 and Hep3B cells, and the quantitative result following H2AFY knockdown. (E) Western blot analysis of cell cycle, apoptosis, EMT related molecular markers in HepG2 and Hep3B cells transfected with H2AFY-shRNA or the negative control. (F) STAT3 signaling pathway was significantly enriched in high-H2AFY expression patients. (G) Evaluation of p-STAT3 and STAT3 expression in HepG2 and Hep3B cells after transfecting H2AFY-shRNA ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001.