Microarray analysis provides new insights into the function of apolipoprotein O in HepG2 cell line

Abstract

Background: Apolipoprotein O (apoO) is a new member of the apolipoprotein family. However, data on its physiological functions are limited and inconsistent. Using a microarray expression analysis, this study explored the function of apoO in liver cells.

Methods: HepG2 cells were treated either with oleic acid or tumor necrosis factor-α for 24 h. mRNA and protein expression of apoO were assessed by quantitative real-time PCR (qRT-PCR) and Western blot respectively. An efficient lentiviral siRNA vector targeting the human apoO gene was designed and constructed. The gene expression profile of HepG2 human hepatocellular carcinoma cells transfected with the apoO silencing vector was investigated using a whole-genome oligonucleotide microarray. The expression levels of some altered genes were validated using qRT-PCR.

Results: ApoO expression in HepG2 cells was dramatically affected by lipid and inflammatory stimuli. A total of 282 differentially expressed genes in apoO-silenced HepG2 cells were identified by microarray analysis. These genes included those participating in fatty acid metabolism, such as ACSL4, RGS16, CROT and CYP4F11, and genes participating in the inflammatory response, such as NFKBIZ, TNFSF15, USP2, IL-17, CCL23, NOTCH2, APH-1B and N2N. The gene Uncoupling protein 2 (UCP2), which is involved in both these metabolic pathways, demonstrated significant changes in mRNA level after transfection.

Conclusions: It is likely that apoO participates in fatty acid metabolism and the inflammatory response in HepG2 cells, and UCP2 may act as a mediator between lipid metabolism and inflammation in apoO-silenced HepG2 cells.

Figure 1

ApoO mRNA and protein changes in HepG2 cells pretreated with 1 mmol/L OA for 24 h. A. The relative levels of apoO mRNA were analyzed by qRT-PCR. Data shown are the mean ± S.E.M from experiments repeated in triplicate with three samples per treatment. **p < 0.01 vs. control. B. The relative levels of apoO protein were analyzed by Western blot analysis. β-actin served as a loading control. These experiments were performed three times, and the results of the densitometric analysis and one representative image are shown. * p < 0.05 vs. control.

Figure 2

Levels of ApoO mRNA and protein in HepG2 cells pretreated with 100 ng/mL TNF-α for 24 h. A. The relative levels of apoO mRNA were analyzed by qRT-PCR. Data shown are the mean ± S.E.M from experiments repeated in triplicate with three samples per treatment. **p < 0.01 vs. control. B. The relative levels of apoO protein were analyzed by Western blot analysis. β-actin served as a loading control. These experiments were performed three times, and the results of the densitometric analysis and one representative image are shown. * p < 0.05 vs. control.

Figure 3

Screening for an efficient lentiviral vector capable of silencing apoO expression. A. HeLa cells observed by fluorescence microscopy 3 days after infection (magnification, ×200). B. The relative levels of apoO mRNA transcripts were analyzed by qRT-PCR. Data shown are means ± S.E.M from three independent experiments. **p < 0.001 vs. negative controls (NC). C. Effects of apoO silencing were measured using Western blot. NC: cells infected with negative control RNAi; LV1: cells infected with apoO-specific RNAi-1; LV2: cells infected with apoO-specific RNAi-2; LV3: cells infected with apoO-specific RNAi-3. β-actin served as a loading control. These experiments were performed three times, and the results of the densitometric analysis and one representative image are shown. **p < 0.001 vs. negative control (NC).

Figure 4

Down-regulation of apoO expression in HepG2 cells using lentivirus-mediated RNAi. A. HepG2 cells were observed by fluorescence microscopy 3 days after infection (magnification, ×200). B. The relative levels of apoO mRNA were analyzed by qRT-PCR. Data shown are the means ± S.E.M of experiments repeated three times with triplicate samples per treatment. **p < 0.01 vs. NC; ## p < 0.01 vs. control. C. Effects of apoO silencing were measured using Western blots. β-actin served as a loading control. These experiments were performed three times, and the results of the densitometric analysis and one representative image are shown. ** p < 0.01 vs. NC; ## p < 0.01 vs. control.

Figure 5

Differentially expressed genes and verification. A. Hierarchical clustering of differentially expressed genes in the negative control group vs. the interference group. The rows show individual genes, while the columns show individual tissue samples. Red denotes high expression and blue denotes low expression. B. Comparison of the expression levels of genes as fold-changes between the negative control group and the apoO-silenced group by microarray analysis and qRT-PCR. Assays were performed from each RNA sample in triplicate. Data were normalized using GAPDH as an endogenous control for RNA input. Fold-changes for these microRNAs from the microarray and qRT-PCR are shown as means ± S.E.M. (n = 6 for each group).