NFYAv1 is a Tumor-Promoting Transcript Associated with Poor Prognosis of Hepatocellular Carcinoma

Abstract

BACKGROUND Nuclear Transcription Factor Y Subunit Alpha (NFYA), together with NFYB and NFYC, form a sequence-specific heterotrimeric nuclear transcription factor (NFY), but their functional role in hepatocellular carcinoma (HCC) is still unclear. In this study, we explored the association between the NFY subunit genes and the survival of primary hepatocellular carcinoma (HCC) patients in The Cancer Genome Atlas (TCGA). The transcript-specific effect on HCC cell growth was studied. MATERIAL AND METHODS RNA-seq data from the Genotype-Tissue Expression Project (GTEx) and TCGA were analyzed in combination. In vitro cellular and molecular studies were conducted using SK-Hep-1 and Hep3B cells. Pearson's correlation coefficients were calculated to assess correlations. Welch's unpaired t test and one-way ANOVA with post hoc Tukey's multiple comparisons were performed. Kaplan-Meier (K-M) survival curves were assessed by conducting log-rank (Mantel-Cox) test. RESULTS NFYA was the only prognosis-related gene. Among the 2 splicing transcripts of NFYA, the long isoform (NFYAv1, NM_002505.5) but not the short-form (NFYAv2, NM_021705.4) was significantly associated with worse progression-free survival (PFS) (high [n=179] vs low [n=179], HR: 1.657, 95% CI: 1.228-2.235, P<0.001) and disease-specific survival (DSS) (high [n=175] vs low [n=175], HR: 1.986, 95% CI: 1.269-3.108, P<0.001) in HCC patients. GO/KEGG analysis in TCGA confirmed that NFYAv1 and NFYAv2 co-expressed (|Pearson's r|≥0.6) genes in primary HCC patients were enriched in quite different GO/KEGG terms. NFYAv1 knockdown significantly decreased cell viability and increased G0/G1 cell cycle arrest. The shRNA only targeting NFYAv1 had a significantly stronger growth-inhibiting effect than the shRNA targeting both NFYAv1 and NFYAv2. CONCLUSIONS This study showed that NFYAv1 is a tumor-promoting transcript associated with poor prognosis of HCC.

Figure 1. HCC patients with elevated NFYA expression had significantly worse survival

(A–F) K-M analysis for DSS (B, D, F) and PFS (A, C, E) comparison was performed. Patients with primary HCC in TCGA were classified into 2 groups by the median NFYA (A, B), NFYB (C, D) or NFYC (E, F) expression. (G) NFYA protein expression in normal liver and HCC tissues. IHC staining images were obtained from the HPA, from the following links: https://www.proteinatlas.org/ENSG00000001167-NFYA/tissue/liver and https://www.proteinatlas.org/ENSG00000001167-NFYA/pathology/liver+cancer#ihc.

Figure 2. NFYAv1, but not NFYAv2, is associated with poor prognosis in patients with HCC

(A, B) Comparison of NFYAv1 and NFYAv2 isoform expression percentage (A) and relative expression (B) in normal liver tissues in GTEx (n=110) and HCC tissues in TCGA (n=359). (C–F) K-M analysis for PFS (C, D) and DSS (E, F) comparison was performed. Patients with primary HCC in TCGA were classified into 2 groups by the median NFYAv2 (C, E) or NFYAv1 (D, F) expression. The log-rank test was performed to evaluate the survival difference. ***, P<0.001.

Figure 3. GO/KEGG analysis identified distinct pathways of NFYAv1 and NFYAv2 co-expressed genes

(A) A Venn diagram showing the common and unique genes highly correlated (|Pearson’s r≥0.6|) with NFYAv1 and NFYAv2 expression in primary HCC cases in TCGA. (B, C) Bubble plots were generated to show the top enriched terms in GO/KEGG pathway enrichment analysis of genes highly correlated (|Pearson’s r≥0.6|) with NFYAv1 (B) and NFYAv2 (C) expression. P values are represented by colors, while gene counts are represented by bubble size.

Figure 4. Knockdown of NFYAv1 impaired HCC cell growth and cell cycle progression

(A) A schematic chart showing the strategy used to perform single-gene GSEA and a summary of the gene sets associated with high NFYAv1 expression. (B) GSEA plots showing the graphical view of the enrichment score for the mitotic spindle and G2/M checkpoint gene sets associated with high NFYAv1 expression. (C) RT-qPCR (upper panel) and western blot (bottom panel) assays were performed to detect the expression of NFYA at the mRNA and protein levels in SK-Hep-1 and Hep3B cells 48 h after infection with lentiviral shNFYA. (D, E) CCK-8 assays were conducted to measure the viability of SK-Hep-1 and Hep3B cells with or without NFYA knockdown. (F–H) Cell cycle analysis was performed through PI staining and following flow cytometry for SK-Hep-1 (F upper panel and G) and Hep3B (F bottom panel and G) cells after lentiviral shNFYA infection. Quantitative measurements (F, G) of the cell cycle phase were conducted. Data are mean±SD (n=3) of 3 biological repeats. **, P<0.01; ***, P<0.001.