Lyudmila Gulyaeva

Regulatory mechanisms of microRNA expression

MicroRNAs (miRs, miRNAs) are small molecules of 18–22 nucleotides that serve as important regulators of gene expression at the post-transcriptional level. One of the mechanisms through which miRNAs regulate gene expression involves the interaction of their “seed” sequences primarily with 3′-end and more rarely with 5′-end, of mRNA transcribed from target genes. Numerous studies over the past decade have been devoted to quantitative and qualitative assessment of miRNAs expression and have shown remarkable changes in miRNA expression profiles in various diseases. Thus, profiling of miRNA expression can be an important tool for diagnostics and treatment of disease. However, less attention has been paid towards understanding the underlying reasons for changes in miRNA expression, especially in cancer cells. The purpose of this review is to analyze and systematize current data that explains reasons for changes in the expression of miRNAs. The review will cover both transcriptional (changes in gene expression and promoter hypermethylation) and post-transcriptional (changes in miRNA processing) mechanisms of regulation of miRNA expression, as well as effects of endogenous (hormones, cytokines) and exogenous (xenobiotics) compounds on the miRNA expression. The review will summarize the complex multilevel regulation of miRNA expression, in relation to cell type, physiological state of the body and various external factors.

Constitutive androstane receptor (CAR) is a xenosensor and target for therapy

Constitutive androstane receptor (CAR, NR1I3), which is under consideration in this review, is a member of the superfamily of nuclear receptors. However, certain features distinguish CAR from the variety of nuclear receptors. First, this receptor has structural features that allow it to display constitutive activity in the absence of a ligand and to interact in a species-specific manner with a huge number of ligands diverse in chemical structure and origin. Second, recently many researchers are focused on CAR because the significance is increasingly shown of its influence on a variety of physiological functions, such as gluconeogenesis, metabolism of xenobiotics, fatty acids, bilirubin, and bile acids, hormonal regulation, etc. In addition to the fundamental scientific interest, the study of CAR is of practical importance because changes in CAR activity can lead to disorders in physiological processes, which finally can result in changes in pathological states. However, despite intensive studies, many mechanisms are still unclear, which makes it difficult to understand the role of CAR in the overall picture of molecular regulation of physiological processes. This review analyzes the features and diversity of the functions of CAR.

Expression of microRNAs, CYP1A1 and CYP2B1 in the livers and ovaries of female rats treated with DDT and PAHs.

AIMS In this study, we determined the expression level of miRNAs and the induction of CYP1A1 and CYP2B1 in the livers and ovaries of female Wistar rats treated with DDT, benzo[a]pyrene (BP), and 3-methylcholanthrene (MC). This study compared CYP1A/2B induction and miRNA expression levels to cast light on a possible role of miRNA in the tissue-specific induction of CYPs. MAIN METHODS The induction of CYP1A1/2B1 enzymes was detected by ethoxy-, pentoxyresorufin O-dealkylation and Western blot analysis. The CYP1A1/2B1 gene expression was determined by RT-real time PCR. Relative levels of expression for selected in silico miR species were determined by real time PCR with small nuclear U6 RNA employed as a reference gene. KEY FINDINGS After bioinformatic analysis, miR-21, 221, 222, and 429 were chosen as potential post-transcriptional regulators of rat CYP1A and CYP2B. It was shown that miR-21, 221, 222, and 429 expression levels decreased in the liver of DDT-, BP-, and MC-treated rats, whereas increases were observed in CYP1A1 and CYP2B1 mRNA expression levels and protein content, and EROD and PROD activities. Conversely, a tendency for elevated levels of miRNAs in the ovaries of inducer-treated female rats was observed. In the ovaries, a high level of CYP1A1 and CYP2B1 mRNA expression was observed, although protein content and enzyme activity were not visible. SIGNIFICANCE These data suggest a potential involvement of miRNA in the post-transcriptional regulation of CYP1A and CYP2B in the livers and ovaries of chemically induced rats.

Species-specific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD)

AIM The aim of the current study was to investigate the species-specific induction of CYP2B by 2,4,6-tryphenyldioxane-1,3 (TPD) in relation to activation of CAR. MAIN METHODS 7-Pentoxyresorufin O-dealkylase (PROD) activity, RT-PCR, Western blot, Electrophoretic mobility shift assays (EMSA). KEY FINDINGS Phenobarbital-like inducer administration significantly up-regulated CYP2B activity in rat and mouse liver in a species-specific manner, in contrast to the effects on CYP2B in lungs, kidneys and brains. In parallel, Western blot analysis showed that the species-specific increase of PROD in liver is related to the high content of CYP2B: phenobarbital (PB) and TPD increased CYP2B in rat liver, PB and 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) - in mouse liver. The CYP2B protein level was unchanged in the lungs of rats and mice after inducer treatment, whereas it was not detected in the kidney and brain of control and treated animals. The hepatic CYP2B activity in both species paralleled the increase of CYP2B mRNA. A detectable CYP2B mRNA level was measured in the lungs of untreated mice and rats, though it was unchanged during induction. Noninducibility of CYP2B in extrahepatic tissues accompanied an absence of constitutive androstane receptor (CAR) gene expression in these tissues. In liver CYP2B induction paralleled the high level of CAR expression detected by RT-PCR. Moreover, PB, TPD and TCPOBOP treatment stimulated nuclear accumulation of CAR and increased CAR receptor NR1-binding activity in animal liver in a species-specific manner. SIGNIFICANCE We have shown that the increased nuclear accumulation and binding activity of CAR are associated with the species-specific up-regulation of CYP2B by TPD in rat liver.

Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on CYP2B induction in mouse liver

2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective inducer of CYP2В in rats, but not in mice. Several analogs of TPD were synthesized. All substituents were entered into the same position of TPD (R=H, cisTPD and transTPD; R=N(CH(3))(2), transpDMA; R=NO(2), transpNO(2); R=F, transpF; R=OCH(3), transpMeO). The purpose of the present study was to investigate the effect of TPD analogs on CYP2B induction in mouse livers. Among the six test compounds, four (R=-N(CH(3))(2), -NO(2), -F, -OCH(3)) demonstrated a dose-dependent induction of mouse CYP2B. To further characterize the compounds, we determined ED50s using sigmoidal dose-response curves. The dose-response study has shown that all active compounds have similar potencies to induce CYP2B in mouse livers. Western-blot analysis and multiplex RT-PCR have shown that the increase of CYP2B activity in mouse liver is related to the high content of CYP2B proteins and paralleled the increase of cyp2b10 mRNA level. ChIP results have demonstrated that the transcriptional enhancement of cyp2b10 gene in response to compounds is accompanied by the increased recruitment of the constitutive androstane receptor (CAR) to its specific binding site (PBREM) on the target gene. Thus, minor structural changes in TPD cause dramatic changes in its ability to induce mouse CYP2B, and it is likely several TPD analogs act by activation of mouse CAR.

Effect of several analogs of 2,4,6-triphenyldioxane-1,3 on constitutive androstane receptor activation

2,4,6-Triphenyldioxane-1,3 (TPD) is a highly effective species-specific inducer of CYP2В in rats. Several analogs of TPD were synthesized to verify a hypothesis that minor changes in the inducer structure can cause changes in induction abilities (R=H, cisTPD and transTPD; R=N(CH(3))(2), transpDMA; R=NO(2), transpNO(2); R=F, transpF; R=OCH(3), transpMeO). Five of six compounds were able to activate CAR in rat liver. Results of Western-blot and ChIP showed that cisTPD and transTPD, transpDMA, transpNO(2), transpF treatment stimulated nuclear accumulation of CAR and evoked CAR receptor PBREM-binding activity in rat liver. cisTPD, transTPD, transpDMA, transpNO(2) and transpF administration significantly increased total CYP content (1.3-2.5 fold) and the level of PROD (12-20 fold), CYP2B specific activity, whereas transpMeO did not have any effects. Western blot and real-time RT-PCR showed that the increase of PROD in liver is related to the high content of CYP2B proteins and paralleled the increase of CYP2B1 (10-43 fold) and CYP2B2 (8-26 fold) mRNAs. At the same time content of CYP2B proteins and CYP2B1 and CYP2B2 mRNA levels were unchanged in rat liver after transpMeO treatment. The dose-response studies have shown that cisTPD, transpDMA, transpF and transpNO(2) have similar potency, and transTPD is less potent derivative. Moreover, it is likely transTPD act as a partial CAR activator. Thus, our results provide evidence to support the conclusion that the differences of TPD analogs ability to activate CYP2B gene expression can be explained by various interactions with CAR.

The search of CAR, AhR, ESRs binding sites in promoters of intronic and intergenic microRNAs

MicroRNAs (miRNAs) play important roles in the regulation of gene expression at the post-transcriptional level. Many exogenous compounds or xenobiotics may affect microRNA expression. It is a well-established fact that xenobiotics with planar structure like TCDD, benzo(a)pyrene (BP) can bind aryl hydrocarbon receptor (AhR) followed by its nuclear translocation and transcriptional activation of target genes. Another chemically diverse group of xenobiotics including phenobarbital, DDT, can activate the nuclear receptor CAR and in some cases estrogen receptors ESR1 and ESR2. We hypothesized that such chemicals can affect miRNA expression through the activation of AHR, CAR, and ESRs. To prove this statement, we used in silico methods to find DRE, PBEM, ERE potential binding sites for these receptors, respectively. We have predicted AhR, CAR, and ESRs binding sites in 224 rat, 201 mouse, and 232 human promoters of miRNA-coding genes. In addition, we have identified a number of miRNAs with predicted AhR, CAR, and ESRs binding sites that are known as oncogenes and as tumor suppressors. Our results, obtained in silico, open a new strategy for ongoing experimental studies and will contribute to further investigation of epigenetic mechanisms of carcinogenesis.

Expression of hormonal carcinogenesis genes and related regulatory microRNAs in uterus and ovaries of DDT-treated female rats

The insecticide dichlorodiphenyltrichloroethane (DDT) is a nonmutagenic xenobiotic compound able to exert estrogen-like effects resulting in activation of estrogen receptor-α (ERα) followed by changed expression of its downstream target genes. In addition, studies performed over recent years suggest that DDT may also influence expression of microRNAs. However, an impact of DDT on expression of ER, microRNAs, and related target genes has not been fully elucidated. Here, using real-time PCR, we assessed changes in expression of key genes involved in hormonal carcinogenesis as well as potentially related regulatory oncogenic/tumor suppressor microRNAs and their target genes in the uterus and ovaries of female Wistar rats during single and chronic multiple-dose DDT exposure. We found that applying DDT results in altered expression of microRNAs-221, -222, -205, -126a, and -429, their target genes (Pten, Dicer1), as well as genes involved in hormonal carcinogenesis (Esr1, Pgr, Ccnd1, Cyp19a1). Notably, Cyp19a1 expression seems to be also regulated by microRNAs-221, -222, and -205. The data suggest that epigenetic effects induced by DDT as a potential carcinogen may be based on at least two mechanisms: (i) activation of ERα followed by altered expression of the target genes encoding receptor Pgr and Ccnd1 as well as impaired expression of Cyp19a1, affecting, thereby, cell hormone balance; and (ii) changed expression of microRNAs resulting in impaired expression of related target genes including reduced level of Cyp19a1 mRNA.

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.

PTEN negative correlates with miR-181a in tumour tissues of non-obese endometrial cancer patients

The effects of microRNAs on PTEN levels are characteristic for many types of cancer. However, the picture of the correlation between the expression levels of PTEN and its targeting microRNAs in endometrial cancer is not fully presented. Our study investigated and analysed the expression levels of PTEN and PTEN-targeting miR-21, miR-181a, miR-214, miR-301a, and miR-1908 in total of 78 samples, out of which 26 samples were from normal endometrium, whereas the 52 samples were from endometrial cancer samples. Our results demonstrated a high variability of individual endometrial cancer samples in the levels of PTEN. The level of miR-181a showed significant increment in endometrial cancer tissues in comparison with normal endometrium. We did not observe any statistically significant correlation between levels of microRNAs and PTEN in a heterogeneous cohort of patients. At the same time, in samples collected from endometrial cancer patients, it was found out that the relationship between PTEN expression and body mass index had significant positive correlation. Moreover, our data demonstrated that the expression of PTEN was significantly decreased, whereas expression of miR-181a was significantly over-expressed in non-obese compared to obese endometrial cancer patients. Additionally, we observed the relationship between PTEN levels and miR-181a related to the cancerous tissues for non-obese patients was established to be negatively correlated. Our findings suggest that decrease of PTEN via increase of miR-181a may be important contributor to endometrial cancer in non-obese patients.