Bisphenol A exposure leads to specific microRNA alterations in placental cells

Abstract

Exposure to bisphenol A (BPA) has been observed to alter developmental pathways and cell processes, at least in part, through epigenetic mechanisms. This study sought to investigate the effect of BPA on microRNAs (miRNAs) in human placental cells. miRNA microarray was performed following BPA treatment in three immortalized cytotrophoblast cell lines and the results validated using quantitative real-time PCR. For functional analysis, overexpression constructs were stably transfected into cells that were then assayed for changes in proliferation and response to toxicants. Microarray analysis revealed several miRNAs to be significantly altered in response to BPA treatment in two cell lines. Real-time PCR results confirmed that miR-146a was particularly strongly induced and its overexpression in cells led to slower proliferation as well as higher sensitivity to the DNA damaging agent, bleomycin. Overall, these results suggest that BPA can alter miRNA expression in placental cells, a potentially novel mode of BPA toxicity.

Figure 1

Unsupervised hierarchical clustering of BPA-treated (TX) and control (CON) based on all miRNA spotted on the microarray in (B) TCL-1 cells, (C) HTR-8 cells, and (D) 3A cells. Columns represent samples and rows represent individual miRNAs. Colors represent fold change intensity.

Figure 2

Quantitative real time PCR analysis of miR-146a expression in cells treated with increasing doses of BPA for 6-days. Fold change refers to the fold change in expression of selected miRNAs in BPA treated triplicates compared to the average of control triplicates. Values represent the mean ±SE of three individual experimental samples run in triplicate. * P < 0.05, for t-test between control (0) and 25 ng/µl dose ** P < 0.01, for t-test between control (0) and 25 ng/µl dose # P < 0.05, for ANOVA across all treatments

Figure 3

Experiments with 3A cells transfected with miR-146a overexpression construct or scrambled control miRNA. (A) Quantitative real time PCR showing expression of miR-146a transfected cells and non-transfected parental 3A cells. (B) Cell proliferation assay showing corrected absorbance at 24, 48 and 72 hours after cell attachment to culture dish. (C) Clonogenic survival of transfected cells following exposure to increasing doses of BPA or (D) bleomycin. * P < 0.01 ** P < 0.001