Gene expression alterations of human liver cancer cells following borax exposure

Abstract

Borax is a boron compound that is becoming widely recognized for its biological effects, including lipid peroxidation, cytotoxicity, genotoxicity, antioxidant activity and potential therapeutic benefits. However, it remains unknown whether exposure of human liver cancer (HepG2) cells to borax affects the gene expression of these cells. HepG2 cells were treated with 4 mM borax for either 2 or 24 h. Gene expression analysis was performed using Affymetrix GeneChip Human Gene 2.0 ST Arrays, which was followed by gene ontology analysis and pathway analysis. The clustering result was validated using reverse transcription‑quantitative polymerase chain reaction. A cell proliferation assay was performed using Celigo Image Cytometer Instrumentation. Following this, 2‑ or 24‑h exposure to borax significantly altered the expression level of a number of genes in HepG2 cells, specifically 530 genes (384 upregulated and 146 downregulated) or 1,763 genes (1,044 upregulated and 719 downregulated) compared with the control group, respectively (≥2‑fold; P<0.05). Twenty downregulated genes were abundantly expressed in HepG2 cells under normal conditions. Furthermore, the growth of HepG2 cells was inhibited through the downregulation of PRUNE1, NBPF1, PPcaspase‑1, UPF2 and MBTPS1 (≥1.5‑fold, P<0.05). The dysregulated genes potentially serve important roles in various biological processes, including the inflammation response, stress response, cellular growth, proliferation, apoptosis and tumorigenesis/oncolysis.

Figure 1.

Upregulated and downregulated genes following treatment with 4 mM borax in HepG2 cells after 2 and 24 h were determined using gene microarray analysis (2 and 24 h groups vs. control group, P<0.05), respectively (over 2-fold change).

Figure 2.

Heatmaps of differentially expressed genes due to borax treatments in HepG2 cells for 2 and 24 h (>2-fold change, P<0.05). Red indicates upregulation whereas green indicates downregulation of gene expression relative to control (untreated cells).

Figure 3.

Enriched GO terms according to biological processes, molecular functions, and cellular components. GO terms are ordered by enrichment score with the highest enriched term at the bottom of the list. Differentially expressed transcripts involved in the term (count) P<0.05 with and fold change >2.0 were included. GO, gene ontology.

Figure 4.

Efficiency of adenovirus infection in HepG2 cells. GFP expression was analyzed in HepG2 cells 48 and 72 h post-infection with AdGFP using fluorescence (lower panels) and light (phase-contrast; upper panels) microscopy (magnification, ×100) to determine the optimal transfection rate for subsequent experiments. (A) (48 h) 40% and (B) (72 h) 80% of cells exhibited GFP expression, respectively. AdGFP, adenoviral green fluorescent protein.

Figure 5.

The decreased expression of genes was established following transfection with each shRNA with real-time PCR. *P<0.05, vs. shControl. shRNA, short hairpin RNA.

Figure 6.

HepG2 cells were transfected with RNAiMax and counts of live adherent HepG2 in cell culture using a Celigo cytometer at the time points indicated. (A) GFP expression of cells infected with different AdGFP-iRNA. (B) Graphs indicated the number of viable cells. (C) Graphs indicated cell growth according to fold change [fold change=shControl/experimental group (transfected with RNAiMax) ≥1.5, P<0.05]. Ctrl, non-targeting shRNA, PC, positive control (specific-targeting shRNA); AdGFP, adenoviral green fluorescent protein.

Figure 6.

HepG2 cells were transfected with RNAiMax and counts of live adherent HepG2 in cell culture using a Celigo cytometer at the time points indicated. (A) GFP expression of cells infected with different AdGFP-iRNA. (B) Graphs indicated the number of viable cells. (C) Graphs indicated cell growth according to fold change [fold change=shControl/experimental group (transfected with RNAiMax) ≥1.5, P<0.05]. Ctrl, non-targeting shRNA, PC, positive control (specific-targeting shRNA); AdGFP, adenoviral green fluorescent protein.