Effects of Endocrine Disruptors o, p'-Dichlorodiphenyltrichloroethane, p, p'-Dichlorodiphenyltrichloroethane, and Endosulfan on the Expression of Estradiol-, Progesterone-, and Testosterone-Responsive MicroRNAs and Their Target Genes in MCF-7 Cells

Abstract

Many studies have shown that dichlorodiphenyltrichloroethane (DDT) exposure raises breast cancer risk. Another insecticide with similar properties is endosulfan, which has been actively used in agriculture after DDT prohibition. Previously, we have identified some estradiol-, progesterone-, and testosterone-sensitive microRNAs (miRNAs, miRs). Because DDT and endosulfan have estrogenic, antiandrogenic, and antiprogesterone properties, we hypothesized that these miRNAs are affected by the insecticides. We quantified relative levels of miRNAs and expression levels of their target genes in breast cancer MCF-7 cells treated with p,p'-DDT, o,p'-DDT, or endosulfan. We also quantified miR-19b expression, which, as previously shown, is regulated by estrogen. Here, we observed that miR-19b expression increased in response not only to estradiol but also to testosterone and progesterone. Treatment of MCF-7 cells with p,p'-DDT or endosulfan decreased the protein levels of apoptosis regulators TP53INP1 and APAF1. In cells treated with o,p'-DDT, the TP53INP1 amount decreased after 24 h of incubation, but increased after 48 h of incubation with insecticide. OXTR expression, which is known to be associated with breast carcinogenesis, significantly diminished under the exposure of all insecticides. In cells treated with p,p'-DDT or o,p'-DDT, the observed changes were accompanied by alterations of the levels of hormone-responsive miRNAs: miR-324, miR-190a, miR-190b, miR-27a, miR-193b, and miR-19b.

Keywords: dichlorodiphenyltrichloroethane; endocrine disruptor; endosulfan; hormone receptor; microRNA; organochlorine pesticide.

Figure 1

Changes in relative levels of miRNAs in MDA-MB-231 cells exposed to an insecticide for 48 h. The Y-axis shows the ratio of the miRNA level in the cells treated with a DDT isomer or endosulfan to the miRNA level in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05).

Figure 2

Changes in relative level of miR-19b in MCF-7 cells exposed to estradiol (E2), progesterone (P4) or testosterone. The Y-axis shows the ratio of the miRNA level in the cells treated with a hormone to the miRNA level in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01.

Figure 3

(A) Relative levels of OXTR mRNA and of the miRNA targeting it (miR-378a) in MCF-7 cells exposed to p,p′-DDT. The Y-axis represents the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of OXTR in cells incubated with p,p′-DDT. The numbers under OXTR bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).

Figure 4

(A) Relative levels of OXTR mRNA and of miRNAs targeting it (miR-378a, miR-324, and miR-342) in MCF-7 cells exposed to o,p′-DDT. The Y-axis shows the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of OXTR in cells incubated with o,p′-DDT. The numbers under OXTR bands represent fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present findings from one of three independent experiments (which yielded similar results).

Figure 5

(A) Relative levels of TP53INP1 mRNA and of miRNAs targeting it (miR-19b, miR-27a, and miR-190a) in MCF-7 cells exposed to p,p′-DDT. The Y-axis indicates the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of TP53INP1 in cells incubated with p,p′-DDT. The numbers under TP53INP1 bands represent fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).

Figure 6

(A) Relative levels of TP53INP1 mRNA and of miRNAs targeting it (miR-193b and miR-342) in MCF-7 cells exposed to o,p′-DDT. The Y-axis shows the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05). (B) Western blot analysis of TP53INP1 in cells incubated with o,p′-DDT. The numbers under TP53INP1 bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present findings from one of three independent experiments (which yielded similar results).

Figure 7

(A) Relative level of miR-190a in MCF-7 cells exposed to o,p′-DDT for 48 h. The Y-axis shows the ratio of the miRNA amount in the cells treated with a DDT isomer to the miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of TP53INP1 in cells incubated with o,p′-DDT. The numbers under TP53INP1 bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present findings from one of three independent experiments (which yielded similar results).

Figure 8

(A) Relative levels of APAF1 mRNA and of miRNAs targeting it (miR-193b and miR-342) in MCF-7 cells exposed to p,p′-DDT. The Y-axis represents the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of APAF1 in cells incubated with p,p′-DDT. The numbers under APAF1 bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).

Figure 9

(A) Relative levels of APAF1 mRNA and of miRNAs targeting it (miR-324, miR-378a, and miR-190b) in MCF-7 cells exposed to o,p′-DDT. The Y-axis represents the ratio of the mRNA or miRNA amount in the cells treated with a DDT isomer to the mRNA or miRNA amount in the cells treated with DMSO. Each value is the mean of three independent experiments. * Significant differences from the control (p < 0.05); ** p < 0.01. (B) Western blot analysis of APAF1 in cells incubated with o,p′-DDT. The numbers under APAF1 bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).

Figure 10

Western blot analysis of PTPRS in cells incubated with p,p′-DDT and o,p′-DDT. The numbers under PTPRS bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results). The levels of proteins APAF1 and OXTR declined in the cells treated with endosulfan for 24 h, and TP53INP1, APAF1, and OXTR levels decreased in the cells treated for 48 h (Figure 11).

Figure 11

Western blot analysis of OXTR, APAF1, and TP53INP1 in cells incubated with endosulfan for 24 and 48 h. The numbers under bands denote fold change in the protein level as compared to the first band (control); each data point was normalized to the corresponding GAPDH band. We present data from one of three independent experiments (which yielded similar results).