Regulation of p53-targeting microRNAs by polycyclic aromatic hydrocarbons: Implications in the etiology of multiple myeloma

Abstract

Multiple myeloma (MM) is a common and deadly cancer of blood plasma cells. A unique feature of MM is the extremely low somatic mutation rate of the p53 tumor suppressor gene, in sharp contrast with about half of all human cancers where this gene is frequently mutated. Eleven miRNAs have been reported to repress p53 through direct interaction with the 3' untranslated region. The expression of nine of them is higher in MM plasma cells than in healthy donor counterparts, suggesting that miRNA overexpression is responsible for p53 inactivation in MM. Here, we report that the environmental carcinogen benzo[a]pyrene (BaP) upregulated the expression of seven p53-targeting miRNAs (miR-25, miR-15a, miR-16, miR-92, miR-125b, miR-141, and miR-200a), while 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (TCDD) upregulated two of them (miR-25 and miR-92) in MM cells. The miR-25 promoter was activated by both BaP and TCDD, and this response was mediated by the aryl hydrocarbon receptor (AhR). We screened 727 compounds that inhibit MM cell survival and down-regulate the expression of p53-targeting miRNAs. We found that (-)-epigallocatechin-3-gallate (EGCG), a constituent of green tea and a major component of the botanical drug Polyphenon® E, reduced the expression of four p53-targeting miRNAs, including miR-25, miR-92, miR-141, and miR-200a. Collectively, these data implicate polycyclic aromatic hydrocarbons and AhR in the regulation of p53-targeting miRNAs in MM and identify a potential therapeutic and preventive agent to combat this deadly disease.

Keywords: microRNAs; multiple myeloma; p53; polycyclic aromatic hydrocarbons; toxicants.

Figure 1

miRNA expression quantification using qPCR in MM1.s cells treated with BaP or TCDD. (A) Schematic representation of gene structure of miR-25 cluster and its orthologs. (B) miRNA sequences of the three miRNA clusters. (C) miRNA expression in MM1.s treated with BaP or TCDD. miRNAs that have the same seed sequence (bold) are grouped together and color-coded.

Figure 2

The promoter of miR-25 contains an AhRE site. (A) Mutagenesis analysis of miR-25 promoter. (B) A reporter assay in HeLa cells demonstrated that BaP and TCDD increased the promoter activities. (C) Inhibition of miR-25 induction in MM1.s cells by TCDD or BaP treatment when AhR or ARNT was knocked down. MM1.s cells were transfected with siAhR or siARNT and 24 hrs later, cells were treated with TCDD (10nM) or BaP (3μM) and miR-25/AhR/ARNT expression was determine after another 24 hrs. Y axis indicates the relative levels of miR-25 or AhR/ARNT mRNA levels with values normalized to that from cells transfected with a siRNA negative control (siControl) and treated with TCDD/BaP. “V” denotes a vehicle control (transfected with siControl and treated with DMSO).

Figure 3

Screening small molecules that inhibit miR-25 expression in MM cells. (A) Cell proliferation of MM1.s treated with 721 compounds at 1.0 μM final concentration. (B) qPCR to measure the levels of miR-25 and p53 mRNA in treated MM1.s cells. (C) Western blotting to measure the p53 protein levels in treated MM1.s cells.

Figure 4

EGCG inhibits the expression of miR-25. (A) qPCR to measure the expression of miRNAs in MM1.s treated with 1.0μM EGCG (B) Reporter assay of the miR-25 promoter in HeLa cells treated with EGCG (C) miR-25 expression in MM1.s cells treated with BaP (3.0 μM) /TCDD (10 nM) and/or EGCG (5.0 μM). Y axis indicates the relative levels of miR-25 in MM cells with treatments normalized to that with mock treatment.

Figure 5

Schematic representation of the potential role of BaP and miRNA overexpression in MM pathogenesis and that of EGCG in MM prevention and intervention.