Transcriptomic analyses of RNA-binding proteins reveal eIF3c promotes cell proliferation in hepatocellular carcinoma

Abstract

RNA-binding proteins (RBPs) play fundamental roles in the RNA life cycle. The aberrant expression of RBPs is often observed in human disease, including cancer. In this study, we screened for the expression levels of 1542 human RBPs in The Cancer Genome Atlas liver hepatocellular carcinoma samples and found 92 consistently upregulated RBP genes in HCC compared with normal samples. Additionally, we undertook a Kaplan-Meier analysis and found that high expression of 15 RBP genes was associated with poor prognosis in patients with HCC. Furthermore, we found that eIF3c promotes HCC cell proliferation in vitro as well as tumorigenicity in vivo. Gene Set Enrichment Analysis showed that high eIF3c expression is positively associated with KRAS, vascular endothelial growth factor, and Hedgehog signaling pathways, all of which are closely associated with specific cancer-related gene sets. Our study provides the basis for further investigation of the molecular mechanism by which eIF3c promotes the development and progression of HCC.

Keywords: Cell proliferation; RNA binding-proteins; eukaryotic translation initiation factor 3 subunit C; hepatocellular carcinoma; the Cancer Genome Atlas.

Figure 1

Transcriptomic analyses of differentially expressed RNA‐binding protein genes in The Cancer Genome Atlas. (a) Clustering heatmap for gene expression of 310 tumor samples and 50 normal samples from The Cancer Genome Atlas liver hepatocellular carcinoma cohort, with rows representing genes and columns representing samples. (b) Track 1 shows 92 upregulated (red boxes) and 19 downregulated (green boxes) RNA‐binding protein genes in liver hepatocellular carcinoma samples. P‐values on track 2 were generated from the survival analysis. Track 3 shows the fold‐change of differentially expressed genes, and track 4 shows somatic mutation frequency. Genes whose expression levels were significantly associated with overall patient survival are shown in red.

Figure 2

Differential eIF3c expression implicates prognostic significance in liver hepatocellular carcinoma (LIHC) samples. (a) Survival curves of 15 upregulated RNA‐binding protein (RBP) genes (red boxes) and three downregulated RBP genes (green boxes). Kaplan–Meier analysis of the correlation between RBP gene expression and overall patient survival. For each gene, patients were dichotomized into high expression (red lines) and low expression (green lines) groups using the median expression level of the individual genes. Log–rank tests were used to determine statistical significance. (b) eIF3c showed significantly higher expression (P = 1.774e‐06, paired Student's t‐test,) in the tumor samples relative to the paired normal samples from The Cancer Genome Atlas LIHC cohort. (c) eIF3c showed significantly higher expression (P = 0.017, unpaired Student's t‐test) in the poorly differentiated tumor samples from The Cancer Genome Atlas LIHC cohort.

Figure 3

eIF3c promotes hepatocellular carcinoma cell proliferation in vitro and tumor growth in vivo. (a) Proliferation of SMMC‐7721 and SK‐HEP‐1 cells targeted by a pool of three eIF3c siRNAs was measured using a cell counting assay. (b) Colony formation for SMMC‐7721 and SK‐HEP‐1 cells transfected with a pool of three eIF3c siRNAs was measured using a colony formation assay. (c) Proliferation of SMMC‐7721 and Huh‐7 cells overexpressing eIF3c was measured using a cell counting assay. (d) Colony formation results for SMMC‐7721 and Huh‐7 cells overexpressing eIF3c are shown. (e,f) Overexpression of eIF3c increased the volume and weight of xenograft tumors. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.

Figure 4

Gene Set Enrichment Analysis of eIF3c‐activated cancer‐related pathways. (a–c) Genes in the hallmark Kyoto Encyclopedia of Genes and Genomes (KEGG) Hedgehog, KEGG vascular endothelial growth factor (VEGF), and KRAS signaling pathways showed significant enrichment in high‐expression eIF3c versus normal‐expression eIF3c in SMMC‐7721 Lenti‐eIF3c and Lenti‐vector cell lines based on RNA sequencing. The top portion of each panel shows the normalized enrichment scores (NES) for each gene; the bottom portion of the plot indicates the value of the ranking metric moving down the list of ranked genes. (d) Real‐time PCR analysis revealed a significant increase in the expression levels of SMO,WNT5B,DHH, RAC2, PCSK1N, and INMBA in SMMC‐7721 cells infected with Lenti‐eIF3c. *P < 0.05; **P < 0.01; ***P < 0.001. ns, not significant.