Roles of Interferon Regulatory Factor 1 in Tumor Progression and Regression: Two Sides of a Coin.

IRF1 is a transcription factor well known for its role in IFN signaling. Although IRF1 was initially identified for its involvement in inflammatory processes, there is now evidence that it provides a function in carcinogenesis as well. IRF1 has been shown to affect several important antitumor mechanisms, such as induction of apoptosis, cell cycle arrest, remodeling of tumor immune microenvironment, suppression of telomerase activity, suppression of angiogenesis and others. Nevertheless, the opposite effects of IRF1 on tumor growth have also been demonstrated. In particular, the "immune checkpoint" molecule PD-L1, which is responsible for tumor immune evasion, has IRF1 as a major transcriptional regulator. These and several other properties of IRF1, including its proposed association with response and resistance to immunotherapy and several chemotherapeutic drugs, make it a promising object for further research. Numerous mechanisms of IRF1 regulation in cancer have been identified, including genetic, epigenetic, transcriptional, post-transcriptional, and post-translational mechanisms, although their significance for tumor progression remains to be explored. This review will focus on the established tumor-suppressive and tumor-promoting functions of IRF1, as well as the molecular mechanisms of IRF1 regulation identified in various cancers.

Constitutive Androstane Receptor Agonist Initiates Metabolic Activity Required for Hepatocyte Proliferation.

Activation of the constitutive androstane receptor (CAR, NR1I3) by chemical compounds induces liver hyperplasia in rodents. 1,4-Bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP), a mouse CAR agonist, is most often used to study chemically induced liver hyperplasia and hepatocyte proliferation in vivo. TCPOBOP is a potent murine liver chemical mitogen, which induces rapid liver hyperplasia in mice independently of liver injury. In recent years, great amount of data has been accumulated on the transcription program that characterizes the TCPOBOP-induced hepatocyte proliferation. However, there are only few data about the metabolic requirements of hepatocytes that divide upon exposure to xenobiotics. In the present study, we have employed liquid chromatography - mass spectrometry technology combined with statistical analysis to investigate metabolite profile of small biomolecules, in order to identify key metabolic changes in the male mouse liver tissue after TCPOBOP administration. Analysis of biochemical pathways of the differentially affected metabolites in the mouse liver demonstrated significant TCPOBOP-mediated enrichment of several processes including those associated with nucleotide metabolism, amino acid metabolism, and energy substrate metabolism. Our findings provide evidence to support the conclusion that the CAR agonist, TCPOBOP, initiates an intracellular program that promotes global coordinated metabolic activities required for hepatocyte proliferation. Our metabolic data might provide novel insight into the biological mechanisms that occur during the TCPOBOP-induced hepatocyte proliferation in mice.

Tumor suppressor PTEN regulation by tobacco smoke in lung squamous-cell carcinoma based on bioinformatics analysis.

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), is a tumor suppressor inactivated in a variety of human cancers. PTEN alteration correlates with lung squamous-cell carcinoma (LUSC) histology. However, it is still unclear how tobacco smoke regulates PTEN in LUSC tissues. In this study, we used free online databases and online tools to analyze PTEN expression and the role of smoking on PTEN alteration in patients with LUSC. We validated bioinformatics data by performing RT-PCR analysis using LUSC patient samples. Our results showed a correlation between the downregulation of PTEN in LUSC tissues compared to normal tissues and smoking exposure. In silico results using online platforms suggest that hsa-mir-301a down-regulates PTEN expression level in smoking patients with LUSC. RT-PCR analysis demonstrated that the PTEN expression was significantly decreased, whereas expression of hsa-mir-301a was up-regulated in the smoker cohort of LUSC tissue compared to adjacent non-cancerous tissues. A significant negative correlation between PTEN and hsa-mir-301a levels was observed in tumour tissues in our cohort of LUSC patients. Our results suggest that the downregulation PTEN gene caused by tobacco smoke-mediated increase of hsa-mir-301a may play an important role in LUSC tumorigenesis.

Genetic Analysis of Multiple Primary Malignant Tumors in Women with Breast and Ovarian Cancer.

Familial cancer syndromes, which are commonly caused by germline mutations in oncogenes and tumor suppressor genes, are generally considered to be the cause of primary multiple malignant neoplasias (PMMNs). Using targeted genomic sequencing, we screened for eight germline mutations: BRCA1 185delAG, BRCA1 T300G, BRCA1 2080delA, BRCA1 4153delA, BRCA1 5382insC, BRCA2 6174delT, CHEK2 1100delC, and BLM C1642T, which provoke the majority of cases of hereditary breast and ovary cancer syndrome (HBOC), in genomic (blood) DNA from 60 women with PMMNs, including breast (BC) and/or ovarian cancer(s) (OC). Pathogenic allelic forms were discovered in nine samples: in seven instances, it was BRCA1 5382insC, and in the following two, BRCA1 4153delA and BRCA1 T300G. The age of onset in these patients (46.8 years) was younger than in the general Russian population (61.0) for BC but was not for OC: 58.3 and 59.4, correspondingly. There were invasive breast carcinomas of no special type and invasive serous ovarian carcinomas in all cases. Two or more tumors of HBOC-spectrum were only in five out of nine families of mutation carriers. Nevertheless, every mutation carrier has relatives who have developed malignant tumors.

Role of Tumor Suppressor PTEN and Its Regulation in Malignant Transformation of Endometrium.

Tumor-suppressive effects of PTEN are well-known, but modern evidence suggest that they are not limited to its ability to inhibit pro-oncogenic PI3K/AKT signaling pathway. Features of PTEN structure facilitate its interaction with substrates of different nature and display its activity in various ways both in the cytoplasm and in cell nuclei, which makes it possible to take a broader look at its ability to suppress tumor growth. The possible mechanisms of the loss of PTEN effects are also diverse - PTEN can be regulated at many levels, leading to change in the protein activity or its amount in the cell, while their significance for the development of malignant tumors has yet to be studied. Here we summarize the current data on the PTEN structure, its functions and changes in its regulatory mechanisms during malignant transformation of the cells, focusing on one of the most sensitive to the loss of PTEN types of malignant tumors - endometrial cancer.

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.

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.

Expression of Estrogen Receptor- and Progesterone Receptor-Regulating MicroRNAs in Breast Cancer.

In ~70% of breast cancer (BC) cases, estrogen and progesterone receptors (ER and PR) are overexpressed, which can change during tumor progression. Expression changes of these receptors during cancer initiation and progression can be caused by alterations in microRNA (miR, miRNA) expression. To assess the association of BC progression with aberrant expression of miRNAs that target ER and PR mRNAs, we quantified miR-19b, -222, -22, -378a, and -181a in BC samples (n = 174) by real-time PCR. Underexpression of miR-222 and miR-378a in stage T2-T4 BC was characteristic for HER2-overexpressing tumors. In addition, the expression of miR-181a and miR-378a was higher in these tumors than in tumors with a HER2 IHC score of 0 or 1+. In tumors with a Ki-67 index ≥ 14%, all tested miRNAs were underexpressed in BC with a high Allred PR score (6-8). In ER-and-PR-negative tumors, miR-22, miR-222, miR-181a, and miR-378a underexpression was associated with Ki-67 index > 35% (median value). MiR-19b and miR-22 underexpression could be a marker of lymph node metastasis in ER- and/or PR-positive tumors with HER2 IHC score 0. Thus, the association of miR-19b, miR-22, miR-222, miR-378a, and miR-181a levels with BC characteristics is influenced by the status of tumor ER, PR, HER2, and Ki-67.

Expression of estrogen-, progesterone-, and androgen-responsive genes in MCF-7 and MDA-MB-231 cells treated with o,p'-DDT, p,p'-DDT, or endosulfan.

Endocrine disruptors are a major concern due to their possible association with hormone-dependent carcinogenesis. Some examples of compounds with such properties are organochlorine pesticides (OCPs). OCPs are persistent pollutants with high lipophilicity, long half-life, and bioaccumulation potential. In the past, some of the most commonly used OCPs were dichlorodiphenyltrichloroethane (DDT) and endosulfan. Here, we investigated the effects of o,p'-DDT, p,p'-DDT, and endosulfan and of hormones estradiol, testosterone, and progesterone on the expression of estrogen, progesterone, and androgen receptors (ER, PR, and AR) and of their target genes (KLF4, VEGFA, CCND1, PRLR, CDKN1A, and BCL6) in MCF-7 and MDA-MB-231 cells. The results confirmed that under the action of the insecticides, there are dose- and time-dependent changes in the expression of these receptors and target genes. As corroborated by an experiment with ER, PR, and AR negative MDA-MB-231 cells, the change in the expression of KLF4, VEGFA, CCND1, and PRLR in MCF-7 cells treated with o,p'-DDT and the change in CDKN1A and PRLR expression in MCF-7 cells treated with p,p'-DDT are likely mediated by ER, PR, and AR pathways. In conclusion, we have identified some targets of DDT and endosulfan and confirmed that the effects of insecticides on the expression of these target genes differ for breast cancer cell lines with different receptor statuses.

Associations between the Levels of Estradiol-, Progesterone-, and Testosterone-Sensitive MiRNAs and Main Clinicopathologic Features of Breast Cancer.

Despite the existing advances in the diagnosis and treatment of breast cancer (BC), the search for markers associated with the clinicopathological features of BC is still in demand. MiRNAs (miRs) have potential as markers, since a change in the miRNA expression profile accompanies the initiation and progression of malignant diseases. The receptors for estrogen, androgen, and progesterone (ER, AR, and PR) play an important role in breast carcinogenesis. Therefore, to search for miRNAs that may function as markers in BC, using bioinformatic analysis and the literature data, we selected 13 miRNAs whose promoter regions contain binding sites for ER or AR, or putative binding sites for ER, AR, and PR. We quantified their expression in MCF-7 cells treated with estradiol, progesterone, or testosterone. The levels of miRNAs sensitive to one or more of these hormones were quantified in BC samples (n = 196). We discovered that high expression levels of miR-190b in breast tumor tissue indicate a positive ER status, and miR-423 and miR-200b levels differ between patients with and without HER2 amplification. The miR-193b, -423, -190a, -324, and -200b levels were associated with tumor size or lymph node status in BC patients, but the presence of these associations depended on the status and expression level of ER, PR, HER2, and Ki-67. We also found that miR-21 expression depends on HER2 expression in ER- and/or PR-positive BC. The levels of miRNA were significantly different between HER2 0 and HER2 1+ tumors (p = 0.027), and between HER2 0 and HER2 2+, 3+ tumors (p = 0.005).

Noncanonical Constitutive Androstane Receptor Signaling in Gene Regulation.

The constitutive androstane receptor (CAR, NR1I3) is extremely important for the regulation of many physiological processes, especially xenobiotic (drug) metabolism and transporters. CAR differs from steroid hormone receptors in that it can be activated using structurally unrelated chemicals, both through direct ligand-binding and ligand-independent (indirect) mechanisms. By binding to specific responsive elements on DNA, CAR increases the expression of its target genes encoding drug-metabolizing enzymes and transporters. Therefore, CAR is mainly characterized as a ligand-dependent or ligand-independent transcription factor, and the induction of gene expression is considered the canonical mode of CAR action. Consistent with its central role in xenobiotic metabolism, CAR signaling includes a collection of mechanisms that are employed alongside the core transcriptional machinery of the receptor. These so-called noncanonical CAR pathways allow the receptor to coordinate the regulation of many aspects of cell biology. In this mini-review, we review noncanonical CAR signaling, paying special attention to the role of CAR in energy homeostasis and cell proliferation.

Downregulation of Acat1 by miR-21 may participate in liver fibrosis upon chronic DDT exposure.

The main objective of the present study was to investigate the toxic effect of long-term exposure to DDT (2,2-dichlorodiphenyl-1,1,1-trichloroethane) on rat livers. Female Wistar rats were treated with once-weekly i.p. doses of DDT (10 and 50 mg/kg) for 12 weeks. Histological analysis revealed significant changes in the liver structure, especially at a dose of 50 mg/kg, which consistent with a fibrotic state. Long-term DDT exposure increased micro RNA-21 (miR-21) level and decreased Acetyl-CoA acetyltransferase 1 (Acat1) mRNA and protein levels in a dose-dependent manner. A dual-luciferase reporter assay confirmed the regulation of the rat Acat1 3'-UTR by miR-21. Previous studies have described the involvement of ACAT1 in fibrogenesis; thus, regulation of the Acat1 gene by miR-21 may play a role in DDT exposure-mediated liver fibrosis.

Association between Lymph Node Status and Expression Levels of Androgen Receptor, miR-185, miR-205, and miR-21 in Breast Cancer Subtypes.

Breast cancer is the most commonly diagnosed cancer among women. Difficulties in treating breast cancer are associated with the occurrence of metastases at early stages of disease, leading to its further progression. Recent studies have shown that changes in androgen receptor (AR) and microRNAs' expressions are associated with mammary gland carcinogenesis, in particular, with the formation of metastases. Thus, to identify novel metastatic markers, we evaluated the expression levels of AR; miR-185 and miR-205, both of which have been confirmed to target AR; and miR-21, transcription of which is regulated by AR, in breast cancer samples (n = 89). Here, we show that the molecular subtypes of breast cancer differ in the expression profiles of AR and AR-associated microRNAs. In addition, the expression of AR and these microRNAs may depend on the expression of PR, ER, and HER2 receptors. Our results show that the possibility of using AR and microRNAs as markers depends on the tumor subtype: a decrease in AR expression may be the marker for the presence of lymph node metastases in patients with HER2-positive subtypes of breast cancer, and disturbance of miR-205, miR-185, and miR-21 expressions may be the marker in patients with a luminal B HER2-positive subtype. Cases with metastases in this type of breast cancer are characterized by a higher level of miR-205 and a lower level of miR-185 and miR-21 in tumor tissues compared to nonmetastatic cases. A decrease in the miR-185 level is also associated with lymph node metastasis in luminal B HER2-negative breast cancer. Thus, the expression levels of AR, miR-185, miR-205, and miR-21 can serve as markers to predict cancer spread to the lymph node in luminal B- and HER2-positive subtypes of breast cancer.

EGFR Polymorphism and Survival of NSCLC Patients Treated with TKIs: A Systematic Review and Meta-Analysis.

Tyrosine kinase inhibitor- (TKI-) based therapy revolutionized the overall survival and the quality of life in non-small-cell lung cancer (NSCLC) patients that have epidermal growth factor receptor (EGFR) mutations. However, EGFR is a highly polymorphic and mutation-prone gene, with over 1200 single nucleotide polymorphisms (SNPs). Since the role of EFGR polymorphism on the treatment outcome is still a matter of debate, this research analyzed the available literature data, according to the PRISMA guidelines for meta-analyses. Research includes PubMed, Scopus, ISI Web of Science, and 14 of genome-wide association studies (GWAS) electronic databases in order to provide quantitative assessment of the association between ten investigated EGFR SNPs and the survival of NSCLC patients. The pooled HR and their 95% CI for OS and PFS for different EGFR polymorphisms using a random or fixed effect model based on the calculated heterogeneity between the studies was applied. The longest and the shortest median OSs were reported for the homozygous wild genotype and a variant allele carriers for rs712829 (-216G>T), respectively. Quantitative synthesis in our study shows that out of ten investigated EGFR SNPs (rs11543848, rs11568315, rs11977388, rs2075102, rs2227983, rs2293347, rs4947492, rs712829, rs712830, and rs7809028), only four, namely, rs712829 (-216G>T), rs11568315 (CA repeat), rs2293347 (D994D), and rs4947492, have been reported to affect the outcome of TKI-based NSCLC treatment. Of these, only -216G>T and variable CA repeat polymorphisms have been confirmed by meta-analysis of available data to significantly affect OS and PFS in gefitinib- or erlotinib-treated NSCLC patients.

Adenomyosis: genetics of estrogen metabolism.

Background To analyze the allelic variants of genes of enzymes involved in estrogen metabolism: CYP1A1, CYP1A2, CYP19 and SULT1A1 using polymerase chain reaction-restriction fragment length polymorphism-restriction fragment length polymorphism (PCR-RFLP) analysis of women with histologically confirmed adenomyosis and women without proliferative diseases of pelvic organs was performed. We studied the following polymorphisms: CYP1A1 M1, T264 → C transition in the 3'-noncoding region; CYP1A2*1F, C734 → A transversion in CYP1A2 gene; C → T transition (Arg264Cys) in exon 7 of CYP19; SULT1A1*2, G638 → A transition (Arg213His) in the SULT1A1 gene. Materials and methods The study included 804 patients. Group I (experimental group) consisted of 268 women with adenomyosis. Inclusion criteria were: histological verification of adenomyosis, consent of patients to participate in the study. Group II (control group) - 536 women without proliferative diseases of the uterus. Inclusion criteria were: lack of proliferative processes of the uterus histologically confirmed by ultrasound examination, patient's consent to participate in the study. Results We found the significant association of C allele, T/C and C/C genotypes of the CYP1A1 gene (CYP1A1 M1 polymorphism), A allele, C/A and A/A genotypes of the CYP1A2 gene (CYP1A2*1F polymorphism) and the T allele, C/T and C/C genotypes of the CYP19 (Arg264Cys polymorphism) gene with the risk for adenomyosis. Conclusions Patients with adenomyosis had increased frequency of C allele, T/C and C/C genotypes of the CYP1A1 gene, A allele, C/A and A/A genotypes of the CYP1A2 gene and T allele and C/T and C/C genotypes of the CYP19 gene and, on the contrary, decreased frequency of the mutant allele and heterozygous and mutant homozygous genotype of the CYP1A2 gene compared to women without proliferative diseases of the uterus.

The expression profile of integrin receptors and osteopontin in thyroid malignancies varies depending on the tumor progression rate and presence of BRAF V600E mutation.

Thyroid cancer (TC) is one of the most common malignancy of the human endocrine system. BRAF V600E mutation is the most frequent genetic alteration of papillary carcinoma, the most frequent TC, which effects RAS-RAF-MEK intracellular signaling pathway. These alterations in RAS-RAF-MEK pathway lead to changes in expression levels of cell membrane integrin receptors and their ligand - extracellular matrix protein osteopontin, which in turn increases the metastatic potential of tumor cells. Thus, integrins and their ligand osteopontin can be considered as potential biomarkers of tumor progression and aggressive tumor phenotypes. The aim of the study was to evaluate the expression levels of integrin receptors ITGA2, ITGA3, ITGAV, ITGA6, ITGA9, ITGB1, ITGB3 and their ligands OPNa, OPNb in the thyroid cancer with different BRAF V600E mutation status. METHODS: Thyroid tumor samples of 70 patients obtained during surgical treatment were analyzed. Expression levels of the investigated genes were evaluated by real time RT-PCR. Fluorescent immunohistochemistry (IHC) was used to confirm the PCR results and to estimate the amount of protein levels. For IHC frozen sections were used. BRAF V600E mutation was determined using allele-specific amplification. Nonparametric criteria (Kruskal Wallis, Wilcoxon and Mann-Whitney tests) were used to evaluate group differences. P values of less than 0.05 were considered as statistically significant. RESULTS: A higher gene expression level of ITGA2 (1.9-fold, p = 0.037), ITGA3 (21.1-fold, p = 0.041) and ITGA5 (2.08-fold, p = 0.048) was observed in papillary thyroid cancer (PTC) tissue in comparison with median expression level in control samples (conventionally normal tissue of thyroid gland). These changes were confirmed by IHC (significant changes for α2 integrin). ITGAV expression level was statistically significantly higher in follicular thyroid cancer (FTC) (2.0-fold, p = 0.040). Next, high gene expression levels in tissue samples of lymph node metastases were observed for ITGA5 (2.92-fold, p = 0.015), OPNb (4.36-fold, p = 0.037). For genes ITGA3 (37.48-fold, p = 0.017790), ITGA6 (18.76-fold, p = 0.028921) and ITGA9 (12.52-fold, p = 0.026710) higher expression level was detected in T3-4 tumors (TNM) compared to tumors classified as T1-2. Presence of BRAF V600E mutation was identified in 20 samples of PTC of 40 (50%). A significant increase of the expression level only of ITGA3 (3.1-fold, p = 0. 0422) was observed in BRAF V600E positive samples. Further, changes in expression levels of integrins and osteopontin were assessed in benign and malignant neoplasms. In PTC samples higher expression of ITGA2 (2.8-fold, p = 0.005), ITGA6 (2.11, p = 0.03) and ITGB1 (2.32-fold, p = 0.02) was detected. In FTC expression level of ITGA6 (2.67, p = 0.007) was higher than in benign thyroid nodules. CONCLUSION: Identified changes in expression levels of the studied genes indicate that they could play an important role in tumor progression, and their expression could be affected by the product of mutant BRAF gene. Integrins and their ligand osteopontin might be considered as potential markers in determining prognosis and treatment of TC.

Expression of the miR-190 family is increased under DDT exposure in vivo and in vitro.

A non-genotoxic insecticide dichlorodiphenyltrichloroethane (DDT), can affect mRNA and microRNA levels, however, its precise mechanism of action remains poorly understood. Using in silico methods we found that the rat miR-190 family is potentially regulated by CAR and ER receptors activated by DDT. We showed that exposure to DDT results in a dose- and organ-dependent increase in the expression of miR-190a, -190b in the liver, uterus, ovaries and mammary gland of female Wistar rats. Additionally, we demonstrate a decrease in protein product level of Tp53inp1, the target gene of these microRNAs, in the rat uterus. It is known that miR-190 is probably regulated by ER in humans, thus we measured the level of miR-190a, -190b in primary cultures of malignant and normal human endometrial cells treated with different doses of DDT. We detected an increase in miR-190b level in normal endometrial cells under DDT exposure. Thus, our results indicate that DDT exposure lead to change in the expression of oncogenic miR-190 family and its target gene Tp53inp1 which may be due to activation of CAR and ER.

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.

Increased expression of miR-155 and miR-222 is associated with lymph node positive status.

Identification of prognostic molecular markers of breast cancer is extremely important. The spreading out of the primary breast tumour cells to the lymphatic system is at the forefront of symbolising the first signs of distant organ metastasis. Deregulated genes in breast cancer tissues that spread to lymph nodes may show early predictive molecular markers. In the present study, we selected five microRNAs, which play a key function in the invasion-metastasis cascade. We investigated the levels of microRNAs in 80 paired samples of BC and matched adjoining tissues, and we examined the potential relationships between microRNA levels and positive lymph node status. Our results attest that three microRNAs (miR-21, miR-155, miR-222) were significantly up-regulated, whilst miR-205 was substantially down-regulated in BC tissues in relation to normal adjoining tissues in a heterogeneous patient cohort. The high levels of two microRNAs, miR-155 and miR-222, showed a statistical relation with the positive lymph node status, especially in patients that had triple negative BC. Conversely, miR-155 was substantially down-regulated in tumour tissues of patients who received preoperative neoadjuvant chemotherapy (NAC) compared with tumour tissues of patients without NAC in cohorts sub-classified to lymph node positive status. Our findings show evidence that the miR-155 and the miR-222 can be defined as molecular markers in regards to cancer patients to prognosticate spread to the lymph node. They also showed that the miR-155 could have crucial significances in BC treatment.

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.

Selection and validation of miRNAs as normalizers for profiling expression of microRNAs isolated from thyroid fine needle aspiration smears.

Fine needle aspiration cytology (FNAC) is currently the method of choice for malignancy prediction in thyroid nodules. Nevertheless, in some cases the interpretation of FNAC results may be problematic due to limitations of the method. The expression level of some microRNAs changes with the development of thyroid tumors, and its quantitation can be used to refine the FNAC results. For this quantitation to be reliable, the obtained data must be adequately normalized. Currently, no reference genes are universally recognized for quantitative assessments of microRNAs in thyroid nodules. The aim of the present study was the selection and validation of such reference genes. Expression of 800 microRNAs in 5 paired samples of thyroid surgical material corresponding to different histotypes of tumors was analyzed using Nanostring technology and four of these (hsa-miR-151a-3p, -197-3p, -99a-5p and -214-3p) with the relatively low variation coefficient were selected. The possibility of use of the selected microRNAs and their combination as references was estimated by RT-qPCR on a sampling of cytological smears: benign (n=226), atypia of undetermined significance (n=9), suspicious for follicular neoplasm (n=61), suspicious for malignancy (n=19), medullary thyroid carcinoma (MTC) (n=32), papillary thyroid carcinoma (PTC) (n=54) and non-diagnostic material (ND) (n=34). In order to assess the expression stability of the references, geNorm algorithm was used. The maximum stability was observed for the normalization factor obtained by the combination of all 4 microRNAs. Further validation of the complex normalizer and individual selected microRNAs was performed using 5 different classification methods on 3 groups of FNAC smears from the analyzed batch: benign neoplasms, MTC and PTC. In all cases, the use of the complex classifier resulted in the reduced number of errors. On using the complex microRNA normalizer, the decision-tree method C4.5 makes it possible to distinguish between malignant and benign thyroid neoplasms in cytological smears with high overall accuracy (>91%).