Perfluorobutane sulfonate exposure disrupted human placental cytotrophoblast cell proliferation and invasion involving in dysregulating preeclampsia related genes

Abstract

We reported that maternal PFBS, an emerging pollutant, exposure is positively associated with preeclampsia which can result from aberrant trophoblasts invasion and subsequent placental ischemia. In this study, we investigated the effects of PFBS on trophoblasts proliferation/invasion and signaling pathways. We exposed a human trophoblast line, HTR8/SVneo, to PFBS. Cell viability, proliferation, and cell cycle were evaluated by the MTS assay, Ki-67 staining, and flow cytometry, respectively. We assessed cell migration and invasion with live-cell imaging-based migration assay and matrigel invasion assay, respectively. Signaling pathways were examined by Western blot, RNA-seq, and qPCR. PFBS exposure interrupted cell proliferation and invasion in a dose-dependent manner. PFBS (100 μM) did not cause cell death but instead significant cell proliferation without cell cycle disruption. PFBS (10 and 100 μM) decreased cell migration and invasion, while PFBS (0.1 μM) significantly increased cell invasion but not migration. Further, RNA-seq analysis identified dysregulated HIF-1α target genes that are relevant to cell proliferation/invasion and preeclampsia, while Western Blot data showed the activation of HIF-1α, but not Notch, ERK1/2, (PI3K)AKT, and P38 pathways. PBFS exposure altered trophoblast cell proliferation/invasion which might be mediated by preeclampsia-related genes, suggesting a possible association between prenatal PFBS exposure and adverse placentation.

Keywords: PFBS; invasion; placenta cytotrophoblast; preeclampsia; proliferation.

Fig 1.. PFBS promotes proliferation in HTR-8/SVneo cells in a dose-dependent fashion.

(1A) Cell viability of HTR-8/SVneo cells after PFBS (0, 0.01, 0.1, 1, 10, 100 µM) exposure as measured by MTS assay with an absorbance of 490 nm. (1B) Cell cycle analysis in HTR-8/SVneo cells treated with PFBS (0, 0.01, 0.1, 1, 10, 100 µM) as measured by flow cytometry. (1C) Representative immunofluorescent images showing DAPI, Ki-67, and DAPI+Ki-67 staining for HTR-8/SVneo cells treated with PFBS (0, 0.01, 0.1, 1, 10, 100 µM). (1D) Ratio of Ki-67(+)/DAPI(+) in HTR-8/SVneo cells after PFBS (0, 0.01, 0.1, 1, 10, 100 µM) exposure.

Fig 2.. PFBS reduces cell migration in HTR-8/SVneo cells in a dose-dependent manner.

(2A) Representative videos of wound closure after PFBS (0, 0.01, 0.1, 1, 10, 100 µM) exposure in HTR-8/SVneo cells. (2B) Rate of distance migrated over time (µm/h) across each wound after PFBS exposure (0, 0.01, 0.1, 1, 10, 100 µM) in HTR-8/SVneo cells. Note: The black arrows point to the edge of the initial scratch. The play button is under each image. White bar is 500 μM

Fig 3.. PFBS significantly disrupts HTR-8/SVneo cell invasion in a dose-dependent fashion.

(3A) Images of filters carrying invaded HTR-8/SVneo cells exposed to PFBS (0, 0.01, 0.1, 1, 10, 100 µM) in a Matrigel invasion assay. (3B) Invasion index (relative to control; 0 µM PFBS) after PFBS exposure (0, 0.01, 0.1, 1, 10, 100 µM) in HTR-8/SVneo cells. Note: white bar is 400 μM

Fig 4.. PFBS significantly down-regulates hypoxia induced factor (HIF)-1α protein level in a time and dose-dependent fashion.

(4A) Protein levels of HIF-1α normalized to GAPDH and a representative Western blot image of HIF-1α and GAPDH in HTR-8/SVneo cells exposed to PFBS (100 µM) in 0, 1, 3, 6 h. (4B) Protein levels of HIF-1α normalized to GAPDH and a representative Western blot image of HIF-1α and GAPDH in HTR-8/SVneo cells exposed to PFBS (0, 0.01, 0.1, 1, 10, 100 µM) in 1 h.

Fig 5.. RNA-seq data revealed dysregulated genes by PFBS in HTR8/SVneo cells

A heatmap and clustering of differentially expressed genes in control (0 µM)) and PFBS (100 µM) treated cells. Note: rep is replicate

Fig 6.. qPCR data validated the dysregulation of genes identified by RNA-seq analyses

Gene expression (fold changes) determined by quantitate-PCR analysis to compare the control (0 µM) and PFBS treatments (0.01, 0.1, 1, 10, 100 µM).

Fig 6.. qPCR data validated the dysregulation of genes identified by RNA-seq analyses

Gene expression (fold changes) determined by quantitate-PCR analysis to compare the control (0 µM) and PFBS treatments (0.01, 0.1, 1, 10, 100 µM).

Fig 6.. qPCR data validated the dysregulation of genes identified by RNA-seq analyses

Gene expression (fold changes) determined by quantitate-PCR analysis to compare the control (0 µM) and PFBS treatments (0.01, 0.1, 1, 10, 100 µM).