Transcriptomic analysis reveals the oncogenic role of S6K1 in hepatocellular carcinoma

Abstract

The p70 ribosomal protein S6 kinase 1 (S6K1), a serine/threonine kinase, is commonly overexpressed in a variety of cancers. However, its expression level and functional roles in hepatocellular carcinoma (HCC), which ranks as the third leading cause of cancer-related death worldwide, is still largely unknown. In the current report, we show the in vivo and in vitro overexpression of S6K1 in HCC. In the functional analysis, we demonstrate that S6K1 is required for the proliferation and colony formation abilities in HCC. By using comparative transcriptomic analysis followed by gene ontology enrichment analysis and Ingenuity Pathway Analysis, we find that the depletion of S6K1 can elevate the expression of a cluster of apoptotic genes, tumor suppressor genes and immune responsive genes. Moreover, the knockdown of S6K1 is predicted to reduce the tumorigenicity of HCC through the regulation of hubs of genes including STAT1, HDAC4, CEBPA and ONECUT1. In conclusion, we demonstrate the oncogenic role of S6K1 in HCC, suggesting the possible use of S6K1 as a therapeutic target for HCC treatment.

Keywords: S6K1; hepatocellular carcinoma; proliferation; transcriptome; tumorigenicity.

Figure 1

In vivo and in vitro overexpression of S6K1 in HCC. (A) copy-number-gain of S6K1 in human hepatocellular carcinoma. DNA copy-number variation of TCGA data is obtained from Oncomine (https://www.oncomine.org). (B) Overexpression of S6K1 transcript in a cohort of 80 HCC tumors (T) and paired adjacent non-tumoral livers (NT). (C) Upregulation of S6K1 in HCC cell lines, Hep3B, PLC5 and HepG2 as compared to the normal liver cell line L02. The mRNA expression level of S6K1 was determined by qRT-PCR. Data are presented as the mean ± SD values (***p = 0.0001, *p < 0.05).

Figure 2

Depletion of S6K1 suppresses in vitro proliferation of HCC cells. (A) Knockdown of S6K1 in HCC cell lines. qRT-PCR and western blotting demonstrated the knockdown efficiency of S6K1 in S6K1-depleted Hep3B, PLC5 and HepG2. (B-C) Depletion of S6K1 suppressed in vitro cell proliferation of HCC. The cell proliferation of S6K1-depleted cell line was determined by (B) WST-1 assay and (C) colony formation assay. Data are presented as the mean ± s.e.m. values (*p < 0.05).

Figure 3

Depletion of S6K1 reduces the proliferation and regains the apoptosis in HCC. (A) Gene-annotation enrichment analysis using The Database for Annotation, Visualization and Integrated Discovery (DAVID). Biological processes with p < 0.05 were considered statistically significant. (B) KEGG pathway analysis using DAVID.

Figure 4

Depletion of S6K1 regulates the gene network to suppress the tumor development.

Figure 5

Schematic diagram to summarize the oncogenic role of S6K1 in HCC.

Figure 6

Quantitative PCR analysis validates the result of transcriptome sequencing. The expression of tumor suppressor genes, apoptotic genes and cell proliferative genes were determined in S6K1-depleted HCC cell lines, as compared to shCtrl cells using qPCR. Data are presented as the mean ± s.e.m. values (*p < 0.05) (n = 8).