Transgenerational Transmission of 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Effects in Human Granulosa Cells: The Role of MicroRNAs

Abstract

Endocrine-disrupting chemicals (EDCs) might contribute to the increase in female-specific cancers in Western countries. 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) is considered the "prototypical toxicant" to study EDCs' effects on reproductive health. Epigenetic regulation by small noncoding RNAs (sncRNAs), such as microRNAs (miRNA), is crucial for controlling cancer development. The aim of this study was to analyze transcriptional activity and sncRNA expression changes in the KGN cell line after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD in order to determine whether sncRNAs' deregulation may contribute to transmitting TCDD effects to the subsequent cell generations (day 9 and day 14 after chronic exposure). Using Affymetrix GeneChip miRNA 4.0 arrays, 109 sncRNAs were found to be differentially expressed (fold change < -2 or >2; p-value < 0.05) between cells exposed or not (control) to TCDD for 3 h and 72 h and on day 9 and day 14 after chronic exposure. Ingenuity Pathway Analysis predicted that following the acute and chronic exposure of KGN cells, sncRNAs linked to cellular development, growth and proliferation were downregulated, and those linked to cancer promotion were upregulated on day 9 and day 14. These results indicated that TCDD-induced sncRNA dysregulation may have transgenerational cancer-promoting effects.

Keywords: TCDD; cancers; dioxin; endocrine-disrupting chemicals (EDCs); epigenetics; granulosa cells; miRNA; transgenerational transmission.

Figure 1

Transcripts of aromatase CYPA1A (a) and CYP1B1 (b) in KGN cells exposed to different doses and duration of TCDD (arbitrary units with reference to PGK1 housekeeping gene expression in DMSO KGN cells at each duration). The measures are expressed as mean ± SEM (n = 3); p-value < 0.05 for all different doses and duration of TCDD vs. DMSO.

Figure 2

Exemplary images of KGN cells after 72 h exposure. (a): untreated (DMSO) cells; (b): cells treated with 10 nM of TCDD. Scale bar: 20 μm.

Figure 3

Cell proliferation (trypan blue viability assay) after exposure to DMSO (control) or 10 nM TCDD for 3 h (acute) and 72 h (chronic exposure) and on day 9 and day 14 after chronic exposure end. Data are the mean ± SEM (n = 9); p-value > 0.05 for all time-points. Y axis = Mean Number of Cells (×10⁵ cells/mL).

Figure 4

Induction of CYP1A1 (a) and CYP1B1 (b) in KGN cells after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD or DMSO (control) and on day 9 and day 14 after chronic exposure’s end (i.e., cells never directly exposed to TCDD). Data are the mean ± SEM (n = 4) (arbitrary units relative to the expression of the housekeeping gene PGK1 in control cells). p values are indicated for each condition (Student’s t test).

Figure 5

Supervised hierarchical clustering using the 21 small non-coding RNAs differentially expressed in KGN cells after exposure to 10 nM TCDD for 3 h (n = 3; black) vs. DMSO (control, n = 3; white); underlined, miRNAs selected for RT-qPCR validation.

Figure 6

Supervised hierarchical clustering using the 39 small non-coding RNAs differentially expressed in KGN cells after exposure to 10 nM TCDD for 3 h (n = 3; black) vs. DMSO (control, n = 3; white); underlined, miRNAs selected for RT-qPCR validation.

Figure 7

Supervised hierarchical clustering using the 18 small non-coding RNAs differentially expressed in KGN cells on day 9 after exposure to 10 nM TCDD for 3 h (n = 3; black) vs. DMSO (control, n = 3; white); underlined, miRNAs selected for RT-qPCR validation.

Figure 8

Supervised hierarchical clustering using the 31 small non-coding RNAs differentially expressed in KGN cells on day 14 after exposure to 10 nM TCDD for 3 h (n = 3; black) vs. DMSO (control, n = 3; white); underlined, miRNAs selected for RT-qPCR validation.

Figure 9

Validation of selected miRNAs in KGN cells (a) exposed to 10 nM TCDD for 72 h, (b,c) on day 9 (9 d) after the end of the 72 h exposure, and (d,e) on day 14 (14 d) after the end of the 72 h exposure; p-values were calculated with Student’s t test.

Figure 10

Venn diagram of the 109 small non-coding RNAs differentially expressed at the indicated time-points after exposure to 10 nM TCDD vs. DMSO (control).

Figure 11

Percentage of transcripts included in each signaling pathway that were predicted by IPA to be upregulated (white) or downregulated (black) after exposure to 10 nM TCDD for 3 h or 72 h and on day 9 (9 d) and day 14 (14 d) after the 72 h exposure end (vs. DMSO, control).

Figure 12

Percentage of transcripts in the MicroRNA Biogenesis Signaling Pathway that were predicted by IPA to be upregulated (white) or downregulated (black) after exposure to 10 nM TCDD for 3 h or 72 h and on day 9 (9 d) and day 14 (14 d) after the 72 h exposure end (vs. DMSO, control).

Figure 13

List of the predicted target genes of the indicated miRNAs differentially expressed between TCDD-exposed and DMSO-exposed (control) KGN cells.

Figure 14

Expression analysis by RT-qPCR of candidate target genes of five miRNAs differentially regulated in KGN cells after acute (3 h) and chronic (72 h) exposure to 10 nM TCDD or DMSO (control) and on day 9 and day 14 after chronic exposure end. Data (arbitrary units relative to the expression of the housekeeping gene PGK1 in control cells) are the mean ± SEM (n = 3); p-values were obtained with Student’s t test.

Figure 15

Illustration of the tumor microenvironment pathway generated using QIAGEN’s Ingenuity Pathway Analysis (IPA).