Effect of benzo[a]pyrene on the expression of miR-126, miR-190a and their target genes EGFL7, TP53INP1 and PHLPP1 in primary endometrial cells.

The main topic of this study was to investigate the effect of benzo[a]pyrene (BP) on microRNAs and their target genes expression levels in primary cell cultures from normal and malignant endometrial tissue. MicroRNA-126 (miR-126) and miR-190a were most sensitive to BP treatment. The treatment of both cultures with BP was accompanied by a decrease of miR-126 level and an increase of EGFL7 gene expression level. BP-induced upregulation of miR-190a was detected only in normal cells and it was accompanied with decrease of mRNA levels of TP53INP1 and PHLPP1 genes. Taking into account that BP promoted the proliferation of normal cells and amplified apoptosis of cancer cells, it is possible that miR-190a is involved in general cellular response to BP. The findings of this study indicate that miR-190a and its target genes may be involved in the regulation of cell fate under BP treatment.

MiR-21 regulates the ACAT1 gene in MCF-7 cells.

AIMS: The purpose of the present study was to determine whether miR-21 regulates the human ACAT1 gene. We also assessed whether transfection of MCF-7 cells with miR-21 mimic/inhibitor leads to changes in ACAT1 mRNA/protein levels, cell proliferation rate, or apoptosis. MAIN METHODS: Regulation of ACAT1 3'UTR by miR-21 was evaluated using a dual-luciferase reporter assay. The effect of miR-21 on mRNA/protein levels of ACAT1 and PTEN (confirmed as an important target of miR-21 for comparison) was measured by qPCR/western blot analysis and immunostaining. Proliferation rate was determined by cell counting. Percentage of cells undergoing late apoptosis was determined by staining with Hoechst 33342/propidium iodide. KEY FINDINGS: Dual-luciferase reporter assay confirmed the regulation of ACAT1 3'UTR by miR-21. Furthermore, transfection of MCF-7 cells with miR-21 mimic decreased mRNA and protein levels of ACAT1 and PTEN genes. In contrast, miR-21 inhibition increased the mRNA and protein levels of both genes studied. Finally, we observed an increase in cell proliferation and decrease in the percentage of cells in late apoptosis in MCF-7 cells transfected with miR-21 mimic, whereas transfection with miR-21 inhibitor led to the opposite effect. SIGNIFICANCE: Our data confirm the hypothesis that miR-21 regulates the human ACAT1 gene. As the expression of this microRNA is altered in many types of cancers, the discovery of novel targets for miR-21 is of particular interest for diagnosis and treatment.

[Expression of miR-21 and Its Acat1, Armcx1, and Pten Target Genes in Liver of Female Rats Treated with DDT and Benzo[a]pyrene].

MiR-21 is the most studied cancer-promoting oncomiR, which target numerous tumor suppressor genes associated with proliferation, apoptosis, and invasion. Here we have studied the synthesis of miR-21 and quantified the mRNA and protein levels for miR-21 potential target genes, i.e., Acat1, Armcx1, and Pten, in the livers of female Wistar rats after their treatment with either 1,1-trichloro-2,2-di(4-chlorophenyl)ethane (DDT) or benzo[a]pyrene (BP). The most important finding appears to be the significant decrease in the miR-21 level the day after treatment with DDT with subsequent rebound. These changes are accompanied by an increase and subsequent drop in the levels of mRNAs and proteins of the Acat1, Armcx1, and Pten genes. These observations indicate the involvement of miR-21 in the posttranscriptional regulation of the Acat1, Armcx1, and Pten genes in response to xenobiotics. We hypothesize that the toxic effects of xenobiotics may be indirect and may manifest by inducing epigenetic changes, particularly through the regulation of miRNAs and their target genes.

[Effect of xenobiotics on microRNA expression in rat liver].

Using bioinformatics analysis we selected microRNAs which could bind 3'-UTR-region of cytochrome P450 (CYP) genes. Three microRNA miR-21, -221, -222, their potential targets might be mRNA for CYP1A1, and two microRNA miR-143, miR-152 for CYP2B1 accordingly were selected for experimental verification. Expression level of these microRNAs in rat liver upon benzo(a)pyrene (BP), phenobarbital (PB), and DDT induction was determined using RT-qPCR method. In rats treated by both BP, and DDT the hepatic content of miR-21, -221, -222 significantly demonstrated a 2-3-fold decrease. The decrease in miR expression was accompanied by a considerable (5.5-8.7-fold) increase in the CYP1A1-mediated EROD activity. The expression of miR-143 remained unchanged after the PB treatment, while the expression of miR-152 increased by 2 times, however, the (10.5-fold) increase in PROD activity of CYP2B was much higher. In the DDT-treated liver PROD activity increased by 20 times, the expression of miR-152 didn't change, and the expression of miR-143 increased by 2 times. The bioinformatics analysis of interactions between microRNAs and targets showed that the studied miRs can potentially bind 3'-end of AhR, ESR1, GR, CCND1, PTEN mRNA. Thus, the expression profile of miR-21, -221, -222, -143, -152 might change under the xenobiotics exposure. In silico analysis confirmed, that microRNAs target not only cytochrome P450 mRNA but also other genes, including those involved in hormonal carcinogenesis, they also can be regulated with studied miRs.

Associativity of microRnA levels in blood serum to quantity and functional activity of haemo - and lymphopoiesis cells in experimental breast cancer.

The work purpose was to reveal an existence of an associativity of the microRNA levels in blood serum to quantitative and functional indices of cells haemo - and lymphopoiesis at the experimental breast cancer induced by N -methyl - N- nitrosourea in the remote period after surgery and carrying out neoadjuvant polychemotherapy. At animals there were investigated levels of microRNA-21, microRNA-221, microRNA-222 and microRNA-429 in serum, also investigated quantitative and functional parameters of cells from bone marrow, from lymph of a chest channel and from spleen. Statistically significant distinctions on the microRNA level in blood serum and an existence of interrelations of microRNA levels with quantitative and functional indices of haemo- and lymphopoiesis cells were revealed.

[Tissue-specific expression of hormonal carcinogenesis target genes in rats treated with polycyclic aromatic hydrocarbons].

We have investigated the effect of polycyclic aromatic hydrocarbons (PAHs) on estrogen-metabolizing genes CYP1A1, CYP1B1, CYP19 and ERalpha and cyclin D1 genes, which control of cell division in estrogen-depended tissues. Treatment of rats with benzo(a)pyren (BP) or 3-methylcholantrene (MC) significantly up-regulated CYP1A1, CYP1B1 gene expression in liver, uterus and ovary, whereas alfa-naphthoflavone (alpha-NF) did not have any effect. The high level of aromatase gene (CYP19) expression was detected in ovary only. Treatment of rats with BP or MC significantly down-regulated expression of this gene (15- and 5,5-fold, respectively), whereas alpha-NF did not have any effect. BP produced an increase in ERalpha and cyclin D1 gene expression in rat liver. This effect was not seen with MC and alpha-NF. ERalpha and cyclin D1 mRNA levels were unchanged in uterus of rats after PAHs treatment. On the other hand, BP treatment caused an increase of the ERalpha and cyclin D1 mRNA levels (3,5- and 2,5-fold, respectively) in ovary, whereas MC and alpha-NF did not have any effects. Thus, our results give evidence for tissue-specific effects of PAHs on expression of genes, which participate in hormonal carcinogenesis. Moreover, the fact that BP and MC treatment affects the expression of estrogen-metabolizing genes and genes, which control of cell division, supports the view that PAHs may be one of the causes of endocrine disorder and consequent hormonal carcinogenesis.