Analysis of the HNF4A isoform-regulated transcriptome identifies CCL15 as a downstream target in gastric carcinogenesis

Abstract

Objective: Hepatocyte nuclear factor 4α (HNF4A) has been demonstrated to be an oncogene in gastric cancer (GC). However, the roles of different HNF4A isoforms derived from the 2 different promoters (P1 and P2) and the underlying mechanisms remain obscure.

Methods: The expression and prognostic values of P1- and P2-HNF4A were evaluated in The Cancer Genome Atlas (TCGA) databases and GC tissues. Then, functional assays of P1- and P2-HNF4A were conducted both in vivo and in vitro. High-throughput RNA-seq was employed to profile downstream pathways in P1- and P2-HNF4A-overexpressing GC cells. The expression and gene regulation network of the candidate target genes identified by RNA-seq were characterized based on data mining and functional assays.

Results: HNF4A amplification was a key characteristic of GC in TCGA databases, especially for the intestinal type and early stage. Moreover, P1-HNF4A expression was significantly higher in tumor tissues than in adjacent non-tumor tissues (P < 0.05), but no significant differences were found in P2-HNF4A expression (P > 0.05). High P1-HNF4A expression indicated poor prognoses in GC patients (P < 0.01). Furthermore, P1-HNF4A overexpression significantly promoted SGC7901 and BGC823 cell proliferation, invasion and migration in vitro (P < 0.01). Murine xenograft experiments showed that P1-HNF4A overexpression promoted tumor growth (P < 0.05). Mechanistically, RNA-seq showed that the cytokine-cytokine receptor interactions pathway was mostly enriched in P1-HNF4A-overexpressing GC cells. Finally, chemokine (C-C motif) ligand 15 was identified as a direct target of P1-HNF4A in GC tissues.

Conclusions: P1-HNF4A was the main oncogene during GC progression. The cytokine-cytokine receptor interaction pathway played a pivotal role and may be a promising therapeutic target.

Keywords: CCL15; Gastric cancer; HNF4A; carcinogenesis; transcriptomics.

Figure 1

Expression and prognostic value of P1-HNF4A in gastric cancer (GC). (A) Schematic of the HNF4A gene and its isoforms (P1 and P2). The antigens recognized by isoform-specific (P1 and P2) antibodies are labeled. (B) Representative expression of P1-HNF4A in tumor and adjacent non-tumor tissues of GC patients. Scale bars = 200 μm and 50 μm. (C) H-score of P1-HNF4A in tumor and adjacent non-tumor tissues using tissue microarray and immunohistochemical analyses (*P < 0.05). (D) The prognostic value of P1-HNF4A in GC patients according to negative and positive expression (P < 0.01).

Figure 2

The effects of both P1- and P2-HNF4A on malignant phenotypes of gastric cancer cells in vitro. (A) P1-HNF4A or P2-HNF4A overexpression lentivirus were transfected into SGC7901 and BGC823 cells. Cell proliferation was examined using the CCK8 assay. OD₄₅₀ absorbance was detected every day for a continuous 4 days (**P < 0.01, n.s., nonsignificant, n = 3). (B) The effects of P1- and P2-HNF4A overexpression on SGC7901 and BGC823 cell cycles using a flow cytometry assay (**P < 0.01; n.s., nonsignificant, n = 3). (C) The effects of both P1- and P2-HNF4A overexpression on SGC7901 and BGC823 cell migrations using transwell experiments. Migrated cells were counted using ImageJ (**P < 0.01; n.s., nonsignificant, n = 3) (Crystal Violet staining, 20×). (D) The effects of both P1- and P2-HNF4A overexpression on SGC7901 and BGC823 cell invasion using transwell experiments. Invaded cells were counted by ImageJ (**P < 0.01; n.s., nonsignificant, n = 3).

Figure 3

The roles of both P1- and P2-HNF4A on gastric cancer (GC) progression in vivo using murine xenograft experiments. (A) BALB/c nude mice underwent orthotopic implantation with SGC7901 cells stably transfected by a negative control, P1-HNF4A, and P2-HNF4A overexpression lentiviruses. (B) The tumors were extracted and measured. Tumor volume was calculated using the formula (V = length × width² × 0.5). The results are expressed as the mean ± SEM (*P < 0.05, n.s., nonsignificant). (C) H&E staining of sections from orthotopic specimens. Scar bars = 200 μm and 50 μm). (D) Ki67 staining of section from orthotopic specimens by immunohistochemistry. Scale bars = 200 μm and 50 μm).

Figure 4

RNA-seq screen of target genes and signaling pathways using downstream P1- and P2-HNF4A overexpression in SGC7901 cells. (A) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showing enrichment of the top 20 signaling pathways in P1-HNF4A overexpression compared to the negative control in SGC7901 cells. (B) KEGG pathway analysis showing enrichment of the top 20 signaling pathways in P2-HNF4A overexpression compared to the negative control in SGC7901 cells.

Figure 5

The effects of CCL15 on malignant phenotypes of gastric cancer (GC) cells in vitro. (A) Negative control and sh-CCL15 lentivirus were transfected in MKN45 cells and CCL15 protein expression was detected by Western blot. β-actin was used as internal control. (B) MKN45 cell viability infected with lentivirus expressing shRNAs targeting CCL15 or control shRNA was examined by CCK8, and OD₄₅₀ was detected at the indicated time (**P < 0.01, n = 3). (C, D) Cell migration and invasion was performed using transwell assays. Migrated (C) or invaded (D) cells were counted by ImageJ (*P < 0.05, **P < 0.01, n = 3) (Crystal Violet staining, 20×). (E) The prognostic value of CCL15 in GC patients using tissue microarrays. (F) The prognostic value of CCL15 in GC patients of the Firehose legacy cohort using the cBioPortal database (Z-Score = 2). (G) The prognostic value of CCL15 in GC patients of the PancancerAtlas cohort using the cBioPortal database (Z-Score = 2).

Figure 6

CCL15 is a direct target of P1-HNF4A in gastric cancer. (A) Relative CCL15 mRNA was examined by qRT-PCR in MKN45 cells transfected with the control, P1-HNF4A, and P2-HNF4A overexpression lentiviruses. GAPDH was used as internal control (**P < 0.01, n = 3). (B) CCL15 promoter fragment (˜2,000 bp) from Ensemble was predicted using JASPAR. Reporter genes containing the classical DR1 motifs or mutational sequences were constructed. (C) Negative control (NC) and the HNF4A2 overexpression plasmid were transiently transfected with CCL15 promoter reporter constructs with the DR1 motif or mutation for 24 h, and luciferase activity was assayed thereafter. The promoter activity was expressed as fold-induction (means ± SEM) compared to that of the NC, n = 3. (**P < 0.01). (D) A chromatin immunoprecipitation assay was performed to show the direct binding of HNF4A2 to the CCL15 promoter. M, Marker. (E) Paraffin sections of human gastric cancer tissues and adjacent non-tumor tissues were immunostained with anti-P1-HNF4A and anti-CCL15 antibodies. H&E staining. Scale bars = 200 μm and 50 μm. (F) The percent of CCL15-low and CCL15-high expression cases in P1-HNF4A negative and positive tissues, respectively according staining score (**P < 0.01). (G) The correlation of CCL15 and HNF4A in the GEPIA database. (H) The correlation of CCL15 and HNF4A in the cBioPortal database. (I) The correlation of CCL15 and HNF4A in the LinkedOmics database.