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Turning to peeling in a dermatological sphere is extensively common and has been used for a long time. From the use of single acids moving on to the so-called compound peelings (associations of more than one substance in the same product) and the combined peelings which take advantage of the action of different substances in a synergistic manner (different products are applied sequentially) in order to best guarantee a greater effectiveness of the treatment for the recommended target. Superficial peelings, combined and not, have led to a drastic reduction in the percentage of incidence of adverse events typical of medium and deep peels. Nevertheless, it has been demonstrated that superficial peels bring about a rejuvenating effect through the mechanical stimulation of the Skin Stress Response System (SSRS), system designated to repairing the damaged tissue and restoring of the normal homeostasis. Clinical trials aims to evaluate the effectiveness and safety of the peppermint peel (PMP) medical device in subjects with different ageing expressions both in qualitative terms (different blemishes such as discolouration, fine wrinkles, elastosis, atony and skin inelasticity, laxity, scarce superficial hydration) and in quantitative terms (degree, extension and number of lesions). A non-controlled multi-centric clinical trial was done in 121 subjects. The use protocol calls for a session every 2 weeks for a total of 4 sessions. Subjects were evaluated before each subsequent session at the first and at 2-4-8 weeks of the fourth and last treatment. During the study there were no adverse events. Only a minimal scurfy flaking and a very slight redness were reported. From an effectiveness point of view, the percentage of therapeutic failure, judged with a score equal to or greater than 4 or 5 in Global Aesthetic Improvement Scale (GAIS) scale was 0%. Best score was obtained in subjects ranging in ages between 38 and 57 (2.02) and in women (2.02) years, while the less satisfactory one was obtained in males (2.14). The study has demonstrated that PMP and the proposed protocol are effective and safe to treat subject with skin signs of chrono and photo ageing, thanks to its capabilities of carrying out a mechanic action indicated as a coadjuvant in the treatment of the dermoepidermic revitalisation through chemical exfoliation and hydration.

Pemetrexed has been widely used in patients with advanced non-small cell lung cancer (NSCLC). The clinical relevance of polymorphisms of folate pathway genes for pemetrexed metabolism have not been fully elucidated yet. The aim of this study was to evaluate the expression levels of circulating miR-22, miR-24, and miR-34a, possibly involved in folate pathway, in NSCLC patients treated with pemetrexed compared with healthy controls and to investigate their impact on patient clinical outcomes. A total of 22 consecutive patients with advanced NSCLC, treated with pemetrexed-based chemotherapy and 27 age and sex matched healthy controls were included in this preliminary analysis. miR-22, miR-24, and miR-34a targets were identified by TargetScan 6.2 algorithm, validating the involvement of these microRNAs in folate pathway. MicroRNAs were isolated from whole blood and extracted with miRNAeasy Mini Kit (Qiagen). miRNA profiling was performed using Real-Time PCR. SPSS 17 was used to data analysis. miR-22, miR-24, and miR-34a were found upregulated (P

Background: MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression, angiogenesis and metastasis. Thrombospondin-1 (TSP-1) has been shown to contrast angiogenesis in vivo. TSP-1 expression levels are inversely correlated with tumor vascularity and metastasis in colon cancer. Bio-informatic statistical analysis indicated that TSP-1 is hypothetical target of miR-21, miR-182, overexpressed in CRC, and let-7i which expression is down-regulated in this tumor. In this work we investigated whether TSP-1 expression could be regulated by miR-21, mir-182 and let-7i in HT29 colon cancer cell line. Methods: To investigated whether miR-21, mir-182 and let-7i directly modulates TSP-1 expression, we transfected HT29 cell line with pre-mir21, pre-mir182 and pre-let7i by using siPortNeo FX tranfection agent and after 48h we evaluated TSP-1 mRNA, using Quantitative Real Time-PCR, and intracellular and secreted protein level performed by Western blotting and ELISA. To confirm the modulation of TSP-1 by miRNAs we transfected HT29 cell line with anti-mir to target the mature form of miR-21, miR182 and let-7i. Results: Using Real-Time PCR we did not find any variation of TSP-1 mRNA expression levels after transfection with pre-mir21 in HT29 cell line, but we observed a down-regulation of cytosolic and secreted protein by Western blot and ELISA. In cells transfected with pre-mir182 we did not observe any down-regulation both TSP-1 mRNA and cytosolic and secreted protein. Finally, we did not find any variation of TSP-1 level in cells transfected with let- 7i. Results were confirmed by transfection with anti-mir21, anti- mir182 and anti-let7i and, using the same method, we evaluated TSP-1 expression. Conclusions: Data suggest that mir-182 induces degradation of TSP-1 mRNA in HT29 cell line, whereas mir-21 affects probably by blockage of TSP-1 translation. Let-7i does not seem involved in regulation of TSP-1 expression in HT29 cells. Understanding the molecular mechanism by which miRNAs regulate TSP-1 expression could be used to restore TSP-1 expression to contrast angiogenic events in colon cancer.

BACKGROUND: The authors, after a thorough review of the literature, and on the basis of personal clinical experience, propose a new approach to the resolution of pain symptomology of border-line cephalea (a pathology as much debated as it is diffused). Therefore, they suggest an interdisciplinary therapeutic protocol. METHODS: After a careful examination of the triggering causes of pain symptomology, they examined 24 patients. They were examined at the out-patient clinic of the Institute of Oral and Maxillo-Facial Surgery, Second University of Study of Naples. With the aid of a questionnaire they gathered the anamnestic data; and, after an objective clinical examination the patients were submitted to neurologic examination. Working together with a neurologist, the patients were firstly examined individually and then, based upon differential differences in symptomatology, they were subdivided into three experimental groups in order to facilitate the association of the therapy to the groups and make follow-up easier. RESULTS: The results were encouraging and demonstrated the necessity to study the patients affected by this type of pathology with a pluridisciplinary approach: gnatologic, psychologic, and neurologic. CONCLUSIONS. Therefore, the importance, if not the necessity, of an interdisciplinary approach is underlined. The authors want to emphasize the importance of not focusing on only one aspect of this kind of pathology, but it must be considered in a wider field including more than one discipline.

The angiopoietin-like 4 (ANGPTL4) protein belongs to a superfamily of secreted proteins structurally related to factors modulating angiogenesis known as angiopoietins. At first, ANGPTL4 has been identified as an adipokine exclusively involved in lipid metabolism, because of its prevalent expression in liver and adipose tissue. This protein regulates lipid metabolism by inhibiting lipoprotein lipase (LPL) activity and stimulating lipolysis of white adipose tissue (WAT), resulting in increased levels of plasma triglycerides (TG) and fatty acids. Subsequently, ANGPTL4 has been shown to be involved in several nonmetabolic and metabolic conditions, both physiological and pathological, including angiogenesis and vascular permeability, cell differentiation, tumorigenesis, glucose homoeostasis, lipid metabolism, energy homeostasis, wound healing, inflammation, and redox regulation. The transcriptional regulation of ANGPTL4 can be modulated by several transcription factors, including PPARα, PPARβ/δ, PPARγ, and HIF-1α, and nutritional and hormonal conditions. Several studies showed that high levels of ANGPTL4 are associated with poor prognosis in patients with various solid tumors, suggesting an important role in cancer onset and progression, metastasis, and anoikis resistance. Here, we have discussed the potential role of ANGPTL4 in mediating the cross talk between metabolic syndromes, such as diabetes and obesity, and cancer through regulation of its expression by PPARs.

// Daniele Fanale 1, * , Valeria Amodeo 1, * , Viviana Bazan 1, * , Lavinia Insalaco 1 , Lorena Incorvaia 1 , Nadia Barraco 1 , Marta Castiglia 1 , Sergio Rizzo 1 , Daniele Santini 2 , Antonio Giordano 3 , Sergio Castorina 4, 5, # , Antonio Russo 1, # 1 Department of Surgical, Oncological and Oral Sciences, Section of Medical Oncology, University of Palermo, Palermo, Italy 2 University Campus Bio-Medico, Department of Medical Oncology, Rome, Italy 3 Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA, USA 4 Fondazione Mediterranea “G.B. Morgagni”, Catania, Italy 5 Department of Biomedical and Biotechnological Sciences, University of Catania, Catania, Italy # Co-last authors, these authors contributed equally to this work * These authors contributed equally to this work Correspondence to: Antonio Russo, e-mail: antonio.russo@usa.net Keywords: bone metastasis, breast cancer, microarray analysis, miRNA expression profile, zoledronic acid Received: March 08, 2016 Accepted: March 31, 2016 Published: April 13, 2016 ABSTRACT Zoledronic acid (ZOL), belonging to third generation bisphosphonate family, is a potent inhibitor of osteoclast-mediated bone resorption, widely used to effectively prevent osteolysis in breast cancer patients who develop bone metastases. Low doses of ZOL have been shown to exhibit a direct anticancer role, by inhibiting cell adhesion, invasion, cytoskeleton remodelling and proliferation in MCF-7 breast cancer cells. In order to identify the molecular mechanisms and signaling pathways underlying the anticancer activity exerted by ZOL, we analyzed for the first time the microRNA expression profile in breast cancer cells. A large-scale microarray analysis of 377 miRNAs was performed on MCF7 cells treated with 10 μM ZOL for 24 h compared to untreated cells. Furthermore, the expression of specific ZOL-induced miRNAs was analyzed in MCF-7 and SkBr3 cells through Real-time PCR. Low-dose treatment with ZOL significantly altered expression of 54 miRNAs. Nine upregulated and twelve downregulated miRNAs have been identified after 24 h of treatment. Also, ZOL induced expression of 11 specific miRNAs and silenced expression of 22 miRNAs. MiRNA data analysis revealed the involvement of differentially expressed miRNAs in PI3K/Akt, MAPK, Wnt, TGF-β, Jak-STAT and mTOR signaling pathways, and regulation of actin cytoskeleton. Our results have been shown to be perfectly coherent with the recent findings reported in literature concerning changes in expression of some miRNAs involved in bone metastasis formation, progression, therapy resistance in breast cancer. In conclusion, this data supports the hypothesis that ZOL-induced modification of the miRNA expression profile contributes to the anticancer efficacy of this agent.

BACKGROUND: Recent findings indicate that exosomes released from cancer cells contain microRNAs (miRNAs) that may be delivered to cells of tumor microenvironment. RESULTS: To elucidate whether miRNAs secreted from chronic myelogenous leukemia cells (CML) are shuttled into endothelial cells thus affecting their phenotype, we first analysed miRNAs content in LAMA84 exosomes. Among the 124 miRNAs identified in LAMA84 exosomes, we focused our attention on miR-126 which was found to be over-overexpressed in exosomes compared with producing parental cells. Transfection of LAMA84 with Cy3-labelled miR-126 and co-culture of leukemia cells with endothelial cells (EC) confirmed that miR-126 is shuttled into HUVECs. The treatment of HUVECs with LAMA84 exosomes for 24 hours reduced CXCL12 and VCAM1 expression, both at the mRNA and protein level, and negatively modulated LAMA84 motility and cells adhesion. Transfection in HUVECs of miR-126 inhibitor reversed the decrease of CXCL12 and restored the motility and adhesion of LAMA84 cells while the over-expression of miR-126, showed opposite effects. CONCLUSION: Our results show that the miR-126 shuttled by exosomes is biologically active in the target cells, and support the hypothesis that exosomal miRNAs have an important role in tumor-endothelial crosstalk occurring in the bone marrow microenvironment, potentially affecting disease progression.

International audience; Research on interspecific competitive interactions among insect parasitoids has often been characterized by laboratory studies in which host insects are exposed to female parasitoids of different species in various sequences and combinations. In the last years, an increasing number of studies have investigated interspecific interactions under field and semi-field conditions although just a few number of works focused on egg parasitoids. In this work, we undertook a two-year study to investigate interspecific interactions between Trissolcus basalis (Wollaston) (Hymenoptera: Platygastridae) and Ooencyrtus telenomicida (Vassiliev) (Hymenoptera: Encyrtidae), two egg parasitoids of the pest Nezara viridula (L.) (Heteroptera: Pentatomidae) that co-occur in cultivated crops. Under semi-field (in out-door mesh cages) and field conditions, we investigated: 1) the seasonal occurrence of competing parasitoid species on sentinel egg masses; 2) the impact achieved by competing species on the shared host on naturally laid egg masses; 3) the outcome of intraguild interactions under controlled conditions. Results from sentinel egg masses showed that T. basalis occurs in May and successfully parasitizes hosts until the end of September/beginning of October, whereas O. telenomicida is mainly occurring in July-August. In both years, it was found that T. basalis is predominant. From naturally laid egg masses, results indicated that T. basalis achieves higher impact on the hosts, even in those egg masses which are parasitized by more than one female of different species (= multiparasitism). Results from manipulating intraguild interactions showed that T. basalis achieves higher impact on N. viridula when released alone, but it suffers from competition with O. telenomicida. The ecological factors that play a role in intraguild interactions in the context of biological control perspective are discussed.

Finding new treatments targeting cancer stem cells (CSCs) within a tumor seems to be critical to halt cancer and improve patient survival. Osteosarcoma is an aggressive tumor affecting adolescents, for which there is no second-line chemotherapy. Uncovering new molecular mechanisms underlying the development of osteosarcoma and origin of CSCs is crucial to identify new possible therapeutic strategies. Here, we aimed to characterize genetically and molecularly the human osteosarcoma 3AB-OS CSC line, previously selected from MG63 cells and which proved to have both in vitro and in vivo features of CSCs. Classic cytogenetic studies demonstrated that 3AB-OS cells have hypertriploid karyotype with 71-82 chromosomes. By comparing 3AB-OS CSCs to the parental cells, array CGH, Affymetrix microarray, and TaqMan® Human MicroRNA array analyses identified 49 copy number variations (CNV), 3,512 dysregulated genes and 189 differentially expressed miRNAs. Some of the chromosomal abnormalities and mRNA/miRNA expression profiles appeared to be congruent with those reported in human osteosarcomas. Bioinformatic analyses selected 196 genes and 46 anticorrelated miRNAs involved in carcinogenesis and stemness. For the first time, a predictive network is also described for two miRNA family (let-7/98 and miR-29a,b,c) and their anticorrelated mRNAs (MSTN, CCND2, Lin28B, MEST, HMGA2, and GHR), which may represent new biomarkers for osteosarcoma and may pave the way for the identification of new potential therapeutic targets.

Objectives: The rapid fatality of pancreatic cancer is, in large part, the result of diagnosis at an advanced stage in the majority of patients. Identification of individuals at risk of developing pancreatic adenocarcinoma would be useful to improve the prognosis of this disease. There is presently no biological or genetic indicator allowing the detection of patients at risk. Our main goal was to identify copy number variants (CNVs) common to all patients with sporadic pancreatic cancer. Methods: We analyzed gene CNVs in leukocyte DNA from 31 patients with sporadic pancreatic adenocarcinoma and from 93 matched controls. Genotyping was performed with the use of the GeneChip Human Mapping 500K Array Set (Affymetrix). Results: We identified 431 single nucleotide polymorphism (SNP) probes with abnormal hybridization signal present in the DNA of all 31 patients. Of these SNP probes, 284 corresponded to 3 or more copies and 147 corresponded to 1 or 0 copies. Several cancer-associated genes were amplified in all patients. Conversely, several genes supposed to oppose cancer development were present as single copy. Conclusions: These data suggest that a set of 431 CNVs could be associated with the disease. This set could be useful for early diagnosis.

We investigated intraguild interactions between two egg parasitoids of Nezara viridula (L.) (Heteroptera: Pentatomidae), Ooencyrtus telenomicida (Vassiliev) (Hymenoptera: Encyrtidae) and Trissolcus basalis (Wollaston) (Hymenoptera: Platygastridae), as the former has the potential to be a facultative hyperparasitoid of the latter. We assessed the suitability of N. viridula eggs for the development of O. telenomicida as a function of egg age when they were unparasitized, or had been attacked by T. basalis at different times prior to exposure to O. telenomicida females. Ooencyrtus telenomicida can exploit healthy N. viridula host eggs up to 5 days of age, just prior to the emergence of N. viridula. This window of opportunity can be extended for an additional 6–7 days through interspecific competition or facultative hyperparasitism. While there are minor fitness costs for O. telenomicida as the result of interspecific larval competition, those costs are greater with facultative hyperparasitism. In choice assays O. telenomicida females discriminated between different quality N. viridula eggs, avoiding those where their progeny would have to develop as facultative hyperparasitoids of T. basalis. Results are discussed with respect to the possible effects that the costs of intraguild parasitism might have on biological control programmes.

The obesity hormone leptin has been implicated in breast cancer development. Breast cancer cells express the leptin receptor and are able to synthesize leptin in response to obesity-related stimuli. Furthermore, leptin is a positive regulator of vascular endothelial growth factor (VEGF) and high levels of both proteins are associated with worse prognosis in breast cancer patients. Peroxisome proliferator-activated receptor γ (PPARγ) ligands are therapeutic agents used in patient with Type 2 diabetes and obesity which have recently been studied for their potential anti-tumor effect. Here, we studied if these compounds, ciglitazone and GW1929, can affect the expression of leptin and VEGF in breast cancer cells. In MDA-MB-231 and MCF-7 breast cancer cells, treatment with submolar concentrations of ciglitazone and GW1929 elevated the expression of leptin and VEGF mRNA and protein, and increased cell viability and migration. These effects coincided with increased recruitment of PPARγ to the proximal leptin promoter and decreased association of a transcriptional factor Sp1 with this DNA region.

Interspecific competitions among parasitoids can affect community structures, and, as a consequence at applicative level, biological control programs. For example, competitions can cause local displacement of inferior species or niche separation. However, the coexistence of species attacking the same host is possible when they adopt different strategies to exploit the resource. In this work we evaluated in field and semi-field conditions intraguild interactions between two egg parasitoids, Trissolcus basalis and Ooencyrtus telenomicida exploring egg masses of Nezara viridula. In semi-field trials, pepper plants were covered with mesh net creating small cages with five plants inside. In each cage, egg masses were placed on plants and then T. basalis and O. telenomicida were released individually or simultaneously. After one week egg masses were recollected. The results showed that T. basalis achieves a high parasitism level when released alone but suffers competition by O. telenomicida in case of simultaneous releasing. Field trials were conducted in tomato fields from May to October in two consecutive years, by monitoring 8 sentinel egg masses placed weekly on plants and recollected one week later, and by collecting naturally laid egg masses that were parasitized only by T. basalis, only by O. telenomicida or by both species. The outcomes showed that T. basalis occurs from June to October, whereas O. telenomicida mainly in July and August, and the former achieves a higher impact on the natural host population than the latter, even when multiparasitism occurs. The ecological role of these results is discussed.

Objective: miRNAs are attractive molecules for cancer treatment, including colon rectal cancer (CRC). We investigate on the molecular mechanism by which miR-182 could regulate thrombospondin-1 (TSP-1) expression, a protein down-regulated in CRC and inversely correlated with tumor vascularity and metastasis. Background: MicroRNAs are small non-coding RNAs that regulate the expression of different genes, involved in cancer progression, angiogenesis and metastasis. miR-182, over-expressed in colorectal cancer (CRC), has like predictive target thrombospondin-1 (TSP-1), a protein inversely correlated with tumor vascularity and metastasis that results downregulated in different types of cancer including CRC. Results: We found that TSP-1 increased after transfection with anti-miR-182 and we showed that miR-182 targets TSP-1 3'UTR-mRNA in both cells. Moreover, we observed that anti-miR-182 did not induce significant variation of Egr-1 expression, but affected the nuclear translocation and its binding on tsp-1 promoter in HCT-116. Equally, Sp-1 was slightly increased as total protein, rather we found a nuclear accumulation and its loading on the TSP-1 promoter in HT-29 transfected with anti-miR-182. Conclusion: Our data suggest that miR-182 targets the anti-angiogenic factor TSP-1 and that anti-miR-182 determines an upregulation of TSP-1 expression in colon cancer cells. Moreover, anti-miR-182 exerts a transcriptional regulatory mechanism of tsp-1 modulating Egr-1 and Sp-1 function. Anti-miR-182 could be used to restore TSP-1 expression in order to contrast angiogenic and invasive events in CRC.

Zoledronic acid (ZOL) is the most potent nitrogen-containing bisphosphonate (N-BPs) that strongly binds to bone mineral and acts as a powerful inhibitor of bone resorption, already clinically available for the treatment of patients with osteolytic metastases. Recent data also suggest that ZOL, used in breast cancer, may provide more than just supportive care modifying the course of the disease, though the possible molecular mechanism of action is still unclear. As breast cancer is one of the primary tumours with high propensity to metastasize to the bone, we investigated, for the first time, differential gene expression profile on Michigan Cancer Foundation-7 (MCF-7) breast cancer cells treated with low doses of ZOL (10 lM). Microarrays analysis was used to identify, describe and summarize evidence regarding the molecular basis of actions of ZOL and of their possible direct anti-tumour effects. We validated gene expression results of specific transcripts involved in major cellular process by Real Time and Western Blot analysis and we observed inhibition of proliferation and migration through 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) and Matrigel assay. We then focused on changes in the cytoskeletal components as fibronectin 1 (FN1), actin, and anti angiogenic compounds as transforming growth factor-b1 (TGF-b1) and thrombospondin 1 (THBS1). The up-regulation of these products may have an important role in inhibiting proliferation, invasion and angiogenesis mediated by ZOL.

Intraguild interactions between two egg parasitoids, Trissolcus basalis (Wollaston) and Ooencyrtus telenomicida (Vassiliev) exploring egg masses of the Southern Green Stink Bug Nezara viridula (L.) were investigated in field and semi-field conditions. Field trials were conducted in tomato fields by using sentinel and naturally laid egg masses in 2010 and 2011. Egg parasitoids monitoring, by sentinel egg masses placed weekly on plants during the tomato growing season and recollected one week later, showed that T. basalis occurs from June to October, whereas O. telenomicida occurs mainly in July and August. In the same fields, collections of naturally laid egg masses display different types of parasitized egg masses: only by T. basalis, only by O. telenomicida and by both species. The outcomes showed that T. basalis achieves a higher impact on the host population than O. telenomicida, even when multiparasitism occurs. Semi-field trials were conducted from July to September 2011, using pepper plants covered with mesh net in order to create small cages with five plants inside. In each cage, egg masses were placed on four plants located near to the cage corners and then T. basalis and O. telenomicida were released individually or simultaneously on the central plants. Egg masses were recollected after one week. The results showed that T. basalis achieves a high parasitization level when released alone but suffers competition by O. telenomicida when both species were simultaneously released.

Introduction: miRNAs are noncoding RNAs that target specific mRNA with subsequent regulation of particular genes, implicated in various biological processes. In cancer, miRNAs could show a different expression from normal tissues. miRNAs have a role as oncogenes when they target tumor suppressor genes and similarly they are tumor suppressors when they target oncogenes. Areas covered: In this review, areas covered include the role of miRNAs in cancer diagnosis, prognosis and research for achievement of therapeutic strategies implicating miRNAs in oncology. As biogenesis of miRNAs is fundamental to understand their usefulness, this has also been discussed. Both miRNA expression profiles in cancer tissues and miRNA levels in peripheral blood were studied for improvement in the management of cancer patients. Expert opinion: miRNAs have the potential for better understanding of tumor biology, but could also provide clinical advancement in management and therapy of various malignancies. The possibility of miRNA detection in peripheralblood would allow an eager expansion of their application in variousclinical settings for cancer. The applicability of miRNA expression profiles still needs to be defined

Breast cancer (BC) is a heterogeneous disease that exhibits familial aggregation. Family linkage studies have identified high-penetrance genes, BRCA1, BRCA2, PTEN and TP53, that are responsible for inherited BC syndromes. Moreover, a combination of family-based and population-based approaches indicated that genes involved in DNA repair, such as CHEK2, ATM, BRIP and PALB2, are associated with moderate risk. Therefore, all of these known genes account for only 25% of the familial aggregation cases. Recently, genome wide association studies (GWAS) in BC revealed single nucleotide polymorphisms (SNPs) in five novel genes associated to susceptibility: TNRC9, FGFR2, MAP3K1, H19 and lymphocyte-specific protein 1 (LSP1). The most strongly associated SNP was in intron 2 of the FGFR2 gene that is amplified and overexpressed in 5-10% of BC. rs3803662 of TNRC9 gene has been shown to be the SNP with the strongest association with BC, in particular, this polymorphism seems to be correlated with bone metastases and estrogen receptor positivity. Relevant data indicate that SNP rs889312 in MAP3K1 is correlated with BC susceptibility only in BRCA2 mutation carriers, but is not associated with an increased risk in BRCA1 carriers. Finally, different SNPs in LSP1 and H19 and in minor genes probably were associated with BC risk. New susceptibility allelic variants associated with BC risk were recently discovered including potential causative genes involved in regulation of cell cycle, apoptosis, metabolism and mitochondrial functions. In conclusion, the identification of disease susceptibility loci may lead to a better understanding of the biological mechanism for BC to improve prevention, early detection and treatment.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of different genes, including genes involved in cancer progression. A functional link between hypoxia, a key feature of the tumor microenvironment, and miRNA expression has been documented. We investigated whether and how miR-20b can regulate the expression of vascular endothelial growth factor (VEGF) in MCF-7 breast cancer cells under normoxic and hypoxia-mimicking conditions (CoCl(2) exposure). Using immunoblotting, ELISA, and quantitative real-time PCR, we demonstrated that miR-20b decreased VEGF protein levels at 4 and 24 h following CoCl(2) treatment, and VEGF mRNA at 4 h of treatment. In addition, miR-20b reduced VEGF protein expression in untreated cells. Next, we investigated the molecular mechanism by which pre-miR-20b can affect VEGF transcription, focusing on hypoxia inducible factor 1 (HIF-1) and signal transducer and activator of transcription 3 (STAT3), transcriptional inducers of VEGF and putative targets of miR-20b. Downregulation of VEGF mRNA by miR-20b under a 4 h of CoCl(2) treatment was associated with reduced levels of nuclear HIF-1 alpha subunit and STAT3. Chromatin immunoprecipitation (ChIP) assays revealed that HIF-1 alpha, but not STAT3, was recruited to the VEGF promoter following the 4 h of CoCl(2) treatment. This effect was inhibited by transfection of cells with pre-miR-20b. In addition, using siRNA knockdown, we demonstrated that the presence of STAT3 is necessary for CoCl(2)-mediated HIF-1 alpha nuclear accumulation and recruitment on VEGF promoter. In summary, this report demonstrates, for the first time, that the VEGF expression in breast cancer cells is mediated by HIF-1 and STAT3 in a miR-20b-dependent manner.