StarD7 knockdown modulates ABCG2 expression, cell migration, proliferation, and differentiation of human choriocarcinoma JEG-3 cells

Abstract

Background: StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.

Methodology/principal findings: Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (-26.4% and -41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and (3)H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.

Conclusions/significance: Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.

Figure 1. Effect of StarD7 siRNA on StarD7 mRNA and protein expression in JEG-3 cells.

Cells were transfected with 100, 150 and 200 nM of StarD7.1 (siD7.1) or StarD7.2 (siD7.2) siRNAs and cultured for 48 h. Control cells were transfected with 200 nM of scrambled siRNA (siC). A-The StarD7 expression was determined by real-time quantitative PCR. Results are expressed as StarD7 mRNA expression in StarD7 siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25 ^th–75 ^th% percentiles of at least three independent experiments performed by triplicate. B- StarD7 protein expression was analyzed by western blot. Protein extracts (100 µg/lane) from scrambled siRNA- (siC), StarD7.1 siRNA- (siD7.1) or StarD7.2 siRNA- (siD7.2) transfected cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-StarD7Ct antibody (top) and with the monoclonal anti-α-tubulin antibody (bottom). A representative blot of at least five independent experiments with similar results is shown. The bar graph represents the densitometric quantification of StarD7 protein levels in StarD7 siRNA-transfected cells normalized to α-tubulin of five separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells defined as 1 (median and 25 ^th–75 ^th% percentiles). *p<0.05 compared to scrambled siRNA-transfected cells.

Figure 2. Effect of StarD7 siRNA on ABCG2 mRNA and protein expression in JEG-3 cells.

A- Cells were transfected with 200 nM of StarD7.1 or scrambled siRNAs and cultured for 48 h or 72 h. ABCG2 expression was determined by real-time quantitative PCR. Results are expressed as ABCG2 mRNA expression in StarD7 siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25 ^th–75^th% percentiles of at least three independent experiments performed by triplicate. B- Confocal microscopy images demonstrating membrane expression of ABCG2 protein (red) in scrambled siRNA-treated JEG-3 cells (siC, upper panel) and low or absent ABCG2 immunostaining in StarD7 siRNA-treated cells (siD7, bottom panel). The nuclei were labelled with Hoescht (blue) and merge images are shown on the right. Bar  = 20 µm (×600). C- Cells were transfected with 200 nM of StarD7.1 siRNA or 200 nM of scrambled siRNA and cultured until 96 h. ABCG2 protein expression was analyzed by western blot. Protein extracts (100 µg/lane) from scrambled siRNA- (siC) or StarD7 siRNA-transfected (siD7) cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-ABCG2 (top), anti-StarD7Ct (middle) and with the monoclonal anti-α-tubulin antibodies (bottom). These immunoblots are representative of at least three separate experiments. D- The bar graph represents the densitometric quantification of ABCG2 protein levels in StarD7 siRNA-transfected cells normalized to α-tubulin of at least four separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells (median and 25^th–75^th% percentiles). *p<0.05 compared to scrambled siRNA-transfected cells.

Figure 3. Effect of StarD7 silencing on JEG-3 cell migration.

A- Wound healing assay in JEG-3 cells treated with StarD7.1 (siD7) or scrambled (siC) siRNAs. An open furrow was generated by scratching confluent cells using a pipette tip. Confluency was restored in controls after 24 h. However, in cells treated with StarD7 siRNA, confluency was not restored after 24 h. B- The distance between furrow edges in the scrambled or StarD7 siRNA-treated cells of three independent experiments was measured and presented graphically as percentage of the initial distance (0 h);*p<0.05 compared to scrambled siRNA-transfected cells. C- Transwell In vitro migration assays. Left panels: representative images show cells migrated to the lower chamber after 48 hours (×200). Right panels: Bar graph represents the percentage of cell migration in seven fields of duplicate wells containing StarD7 siRNA-treated cells relative to scrambled siRNA ones (median and 25^th–75^th% percentiles, n = 3); *p<0.05 compared to scrambled siRNA-transfected cells. D-Cell proliferation was determined by BrdU (red) staining of JEG-3 cells treated with scrambled (left panel) or StarD7.1 (right panel) siRNAs. The nuclei were labelled with Hoescht (blue, middle panels) and merge images are shown on the bottom panels. Bar  = 50 µm (×200). The images are representative of three experiments with consistent results.

Figure 4. The de novo biosynthesis of total glycerophospholipids in StarD7 siRNA-treated JEG-3 cells.

A- Relative ³H-glycerophospholipid synthesis in StarD7.1 (siD7) siRNA-treated JEG-3 cells compared to scrambled (siC) siRNA-treated cells defined as 1. Data are median and 25^th–75^th% percentiles (n = 4 independent experiments). *p<0.05 compared to scrambled siRNA-transfected cells. B- Percentage of distribution of major individual ³H-phospholipids in StarD7 siRNA-treated JEG-3 cells or in scrambled siRNA cells. Data are mean ± SEM (n = 2 independent experiments). Phosphatidylcholine (PC) is the species with the highest precursor incorporation in both cell conditions, followed by phosphatidylserine (PS) and after phosphatidylinositol (PI) and phosphatidylethanolamine (PE) with a minor percentage.

Figure 5. βhCG and syncytin-1 mRNA expression are upregulated in JEG-3 cells by StarD7 silencing.

Quantitative RT-PCR analysis of βhCG (A) and syncytin-1 (B) mRNAs in siRNAs-treated JEG-3 cells is shown. Analysis was performed using cDNAs derived from one µg of total RNA extracted from cells treated with StarD7.1 (siD7) or scrambled (siC) siRNA and cultured until 72 hours. Results are expressed as mRNA expression in StarD7siRNA-transfected cells after normalizing to cyclophilin A relative to the corresponding normalized mRNA levels in scrambled siRNA-transfected cells. The values represent the median and 25^th–75^th% percentiles of triplicate results obtained from at least three independent experiments; *p<0.05 compared to scrambled siRNA-transfected cells.

Figure 6. βhCG protein expression is upregulated in JEG-3 cells by StarD7 silencing.

Cells were transfected with scrambled or StarD7.1 siRNAs for 6 h and then cultured until 72 hours. A- βhCG and StarD7 protein expression were analyzed by western blot. Protein extracts (100 µg/lane) from StarD7.1 siRNA-transfected (siD7) or scrambled siRNA-transfected (siC) cells were electrophoresed on a 7.5% SDS polyacrylamide gel and transferred to a nitrocellulose filter. Filters were incubated with anti-βhCG (top), anti-StarD7Ct (middle) and with the monoclonal anti-α-tubulin antibodies (bottom). These immunoblots are representative of at least three separate experiments. B- The bar graph represents the densitometric quantification of βhCG protein levels in StarD7 siRNA-transfected JEG-3 cells normalized to α-tubulin of five separate experiments relative to the corresponding normalized protein levels in scrambled siRNA-transfected cells defined as 1 (median and 25^th–75^th% percentiles). C- hCG secretion from JEG-3 cells after 48 h or 72 h of culture in StarD7.1 siRNA-transfected condition compared to scrambled siRNA-transfected cell culture defined as 1 (n = 5). *p<0.05 compared to scrambled siRNA-transfected cells.

Figure 7. Effect of StarD7 knockdown on JEG-3 cell differentiation.

Cells were transfected with scrambled (siC) or StarD7.1 (siD7) siRNAs for 6 h and then cultured until 72 hours. A- Detection of desmoplakin (red) in siRNA-treated JEG-3 cells was performed by immunofluorescence (left panel). The nuclei were labelled with Hoescht (blue) and merge images are shown on the right. Syncytial structures were characterized by the absence or incomplete desmoplakin staining (arrows). In control cells, desmoplakin labeling was continuous at the periphery of cells. Bar = 20 µm (×400). B- Percentage fusion was determined in cells transfected with StarD7.1 or scrambled siRNAs and represents the percentage of nuclei number in syncytia. Twenty fields were counted for each condition in three different experiments performed in duplicate as described in Materials and Methods. Results are depicted in terms of median percentage and 25^th–75^th% percentiles; *p<0.05 compared to scrambled siRNA-transfected cells.