Transcriptome Analysis of the Anti-Proliferative Effects of Ginsenoside Rh3 on HCT116 Colorectal Cancer Cells

Abstract

The mechanism of ginsenoside Rh3 activity against cancer remains unclear. This study aimed to investigate the underlying mechanism. The effects of Rh3 on the cell proliferation, migration and invasion, and cycle and apoptosis were analyzed using CCK-8 assay, transwell migration assay and flow cytometry, respectively. The RNA transcriptome was sequenced and data were analyzed by R software. Protein expression and protein-protein interactions were determined by Western blotting and co-immunoprecipitation, respectively. The results showed Rh3 inhibited HCT116 cell proliferation, invasion, and migration, arrested cells at G1 phase; and increased apoptosis. Rh3 downregulated 314 genes and upregulated 371 genes. Gene Set Enrichment Analysis (GSEA) using The Kyoto Encyclopedia of Genes Genomics ranked DNA replication first, while GSEA using Gene Ontology ranked the initiation of DNA replication first. Compared with tumor data from The Cancer Genome Atlas (TCGA), most of genes related to DNA replication were oppositely regulated by Rh3. Furthermore, Rh3 down-regulated key protein expression related to DNA replication (Orc6, Cdt1, and Mcm2), but did not affect the loading of Mcm complexes onto ORC complexes nor the phosphorylation at ser139 of Mcm2. Therefore, Rh3 may inhibit colorectal cancer HCT116 cells by downregulation of genes related to DNA replication.

Keywords: DNA replication; colorectal cancer; ginsenoside (Rh3); proliferation; transcriptome analysis.

Figure 1

Effect of Rh3 on cell viability, migration, invasion, and cell cycle progression in HCT116 cells. (A) Cell proliferation after treatment with different concentration of Rh3 (25, 50, 75, 100, 125, 150 or 200 μg/mL) or the same amount of solvent (DMSO) for 24 h (CCK-8 assay, photo magnification 40×). (B,C) Effect of Rh3 on HCT116 cell migration and invasion (transwell assay, photo magnification 100×). (D,E) Apoptosis and cell cycle analyses after treatment with Rh3 for 24 h (flow cytometry). Groups were compared using a two-tailed Student’s t-test. Error bars: standard deviation, * p < 0.05, ** p < 0.01.

Figure 2

Effect of Rh3 on gene transcription in HCT116 cells. (A) Heatmap of genes with differential expression. Colored bars indicate scaled reads per kilobase of transcript (RPKM) of each gene (blue: high expression in the Rh3 group, red: low expression in the Rh3 group). Dendrograms represent similarities in clusters of samples (left) and in levels of gene expression (top). (B) Volcano plot of differential gene expression. The horizontal dashed line indicates p = 0.05, and the vertical dashed lines indicate log₂FC = +1 and log₂FC = −1. (C,D) GSEA of GO and KEGG pathways with differential gene expression (GSEA version 4.1.0). The first ranks of GSEA of the GO and KEGG pathways are DNA replication initiation and DNA replication pathways; other pathways are shown in Tables S1 and S2. (E) Log₂FC in the expression of specific genes that function in DNA replication in colon cancer tumor tissue relative to adjacent normal colon tissue (blue, from TCGA), and in the Rh3 group relative to the DMSO group (red).

Figure 3

Effect of Rh3 on the expression of DNA replication initiation proteins. (A) Expression of Mcm2, Cdt1, and Orc6 in HCT116 cells (Western blotting with actin as the loading control). (B) Effect of Rh3 on the interaction between Orc6 and Mcm2 (Co-IP assay). (C) Expression of Mcm2 and phosphorylated Mcm2 (western blotting). Groups were compared using a two-tailed Student’s t-test. ** p < 0.01.

Figure 4

Effect of Rh3 on the expression of invasion and migration associated proteins. (A): Expression of ZO-1, N-Cadherin, E-Cadherin, Vimentin, MMP-9, β-Catenin, and LOX in HCT116 cells were detected by Western blotting with actin as the loading control. (B) Gray analysis of western blotting on ZO-1, N-Cadherin, E-Cadherin, Vimentin, MMP-9, LOX, and β-Catenin. Groups were compared using a two-tailed Student’s t-test. ** p < 0.01.