Transcriptome wide functional analysis of HBx expressing human hepatocytes stimulated with endothelial cell cross-talk

Abstract

Identification of genes dysregulated during the hepatitis B virus (HBV)-host cell interaction adds to the understanding of underlying molecular mechanisms and aids in discovering effective therapies to improve prognosis in hepatitis B virus (HBV)-infected individuals. Through bioinformatics analyses of transcriptomics data, this study aimed to identify potential genes involved in the cross-talk of human hepatocytes expressing the HBV viral protein HBx with endothelial cells. Transient transfection of HBV viral gene X (HBx) was performed in THLE2 cells using pcDNA3 constructs. Through mRNA Sequencing (RNA Seq) analysis, differentially expressed genes (DEGs) were identified. THLE2 cells transfected with HBx (THLE2x) were further treated with conditioned medium from cultured human umbilical vein derived endothelial cells (HUVEC-CM). Gene Ontology (GO) enrichment analysis revealed that interferon and cytokine signaling pathways were primarily enriched for the downregulated DEGs in THLE2x cells treated with HUVEC-CM. One significant module was selected following protein-protein interaction (PPI) network generation, and thirteen hub genes were identified from the module. The prognostic values of the hub genes were evaluated using Kaplan-Meier (KM) plotter, and three genes (IRF7, IFIT1, and IFITM1) correlated with poor disease specific survival (DSS) in HCC patients with chronic hepatitis. A comparison of the DEGs identified in HUVEC-stimulated THLE2x cells with four publicly available HBV-related HCC microarray datasets revealed that PLAC8 was consistently downregulated in all four HCC datasets as well as in HUVEC-CM treated THLE2x cells. KM plots revealed that PLAC8 correlated with worse relapse free survival and progression free survival in HCC patients with hepatitis B virus infection. This study provided molecular insights which may help develop a deeper understanding of HBV-host stromal cell interaction and open avenues for future research.

Keywords: Conditioned medium; Endothelial cells; HBV X gene (HBx); HUVEC; Hepatitis B; THLE2; Transcriptome.

Fig. 1. Intracellular expression of HBx gene in THLE2 cells from pcDNA3 constructs.

a. Transfection efficiency was measured to be around 60% by analyzing the percent of GFP positive THLE2 cells co-transfected with pcDNA3-HBx and pEGFP-C3 b. PCR product from THLE2 cells transfected with empty-pcDNA3 and pcDNA3-HBx analysed by gel electrophoresis on 2% agarose. HBx gene (465 bp long) corresponding to 0.5-kb band of marker was successfully amplified from cells transfected with pcDNA3-HBx. c. Graph representing the real time expression of HBx gene in cells transfected with pcDNA3-HBx. d. Western blot representing HBx-specific bands in protein samples from THLE2 cells transfected with pcDNA3-HBx.

Fig. 2. Identification of genes differentially regulated in THLE2x cells compared to control THLE2-pcDNA3 cells.

a. Volcano plot of the total expression of genes in a THLE2x vs THLE2-pcDNA3 group. The x-axis represents the log2 (fold change) values, and the y-axis represents the -xlog10 (adjusted p values) values for gene expression. Each dot represents a gene identified from the RNA-seq data. Red dots indicate 48 up-regulated DEGs, blue dots indicate 100 downregulated DEGs, and black dots indicate nondifferential expressed genes. b. Heatmap of 148 DEGs in the THLE2x cells compared to THLE2-pcDNA3 cells. The output thresholds were |log2FC| > 1 and p-value< 0.05.

Fig. 3. Identification of genes differentially regulated in THLE2x cells treated with HUVEC-CM (THLE2x-CM) compared to THLE2x cells.

a. Volcano plot of the total expression of genes in THLE2x-CM group vs THLE2-x group. The x-axis represents the log2 (fold change) values, and the y-axis represents the -log10 (adjusted p values) values for gene expression. Red dots indicate 74 up-regulated DEGs, blue dots indicate 106 downregulated DEGs, and black dots indicate nondifferential expressed genes. b. Heatmap of 180 DEGs in the THLE2x-CM cells compared to THLE2x cells. The output thresholds were |log2FC| > 1 and p-value< 0.05.

Fig. 4

GO (Gene Ontology) enrichment analysis for DEGs in THLE2x-CM cells compared THLE2x using FunRich Downregulated DEGs in THLE2x cells when treated with HUVEC-CM were enriched in the GO categories “cellular component” (a), “molecular function” (b), and “biological pathways”(c). p values <0.05 were statistically significant.

Fig. 5. Protein-protein interaction (PPI) network and identification of protein clusters with significant modules.

a. PPI network was generated by STRING with up-regulated and downregulated DEGs identified in THLE2x cells treated with HUVEC-CM compared to THLE2x cells. b. One significant module, Cluster 1 (MCODE score = 12) was identified using the molecular complex detection (MCODE) method. c. Protein cluster with MCODE score = 3 was identified as Cluster 2. Red nodes in the protein clusters indicate downregulated genes.

Fig. 6

Relative fold change expression of 13 hub genes by RT-qPCR in THLE2x and HepG2x cells after treatment with HUVEC-CM. Relative fold change in mRNA expression of ISG15, IFITM1, IFI44L, IFIH1, MX1, USP18, IRF7, OAS2, MX2, XAF1, RTP4, IFIT1, and OAS1 analysed in THLE2x cells (a), and HepG2x cells (b) treated with HUVEC-CM compared to non-treated control cells. The expression levels of the genes were normalised to the expression of ACTB. *p < 0.05; **p < 0.01; *** < 0.001; ns, non-significant.

Fig. 7. Kaplan–Meier survival curves of the 13 hub genes.

a-m. Graph representing disease specific survival (DSS) in HCC patients grouped according to low and high expression of ISG15, IFITM1, IFI44L, IFIH1, MX1, USP18, IRF7, OAS2, MX2, XAF1, RTP4, IFIT1, and OAS1 genes. p values <0.05 were considered as statistically significant.

Fig. 8. PLAC8 was differentially downregulated in THLE2x-CM group and in HBV-related HCC datasets.

a-b. Venn diagram showing that PLAC8 gene is downregulated in HBV associated HCC datasets and in THLE2x cells treated by HUVEC CM c-d. Graph representing relapse free survival (RFS), (c), and progression free survival (PFS), (d) in HCC patients grouped according to low and high expression of PLAC8 gene. p values <0.05 were considered as statistically significant.