Dual Suppressive Effect of miR-34a on the FOXM1/eEF2-Kinase Axis Regulates Triple-Negative Breast Cancer Growth and Invasion

Purpose: Recent studies indicated that dysregulation of noncoding KNAs (ncRNA) such as miRNAs is involved in pathogenesis of various human cancers. However, the molecular mechanisms underlying miR-34a are not fully understood in triple-negative breast cancer (TNBC). Experimental Design: We performed in vitro functional assays on TNBC cell lines to investigate the role of mi R-34a in FOLM1/eEF2K signaling axis. TNBC tumor xenograft models were used for in vivo therapeutic delivery of miR-34a. Results: In this study, we investigated the role of p53-driven ncRNA miR-34a and found that miR-34a is associated with significantly longer patient survival in TNBC and inversely correlated with levels of proto-oncogenic eEF2K, which was associated with significantly shorter overall patient survival, We showed that miR-34a directly binds to the 3'-untranslated region of eEF2K and FOXM1 mRNAs and suppresses their expression, leading to inhibition of TNBC cell proliferation, motility, and invasion. Notably, restoring miR-34a expression recapitulated the effects of inhibition of eEF2K and FOXM1, the transcription factor for eEF2K and the direct target of p53, in TNBC cell lines, whereas overexpression of eEF2K and FOXM1 rescued the effects and signaling pathways mediated by miR-34a. Moreover, in vivo therapeutic delivery of miR-34a nanopartides by systemic intravenous administration delayed tumor growth of two different orthotopic TNBC tumor xenograft models by inhibiting eEF2K and FOXM1, intratumoral proliferation and angiogenesis, and inducing apoptosis. Conclusions: Overall, our findings provide new insights into the tumor suppressor role of miR-34a by dual-targeting of FOXM1/eEF2K signaling axis and suggest that miR-34a-based gene therapy may be a potential therapeutic strategy in TNBC. (C)2018 AACR.NIH/NCIUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [R21CA199050, P30CA016672]; noncoding RNA center; NATIONAL CANCER INSTITUTEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Cancer Institute (NCI) [P30CA016672] Funding Source: NIH RePORTERThis work was supported in part by grants from the NIH/NCI (R21CA199050 and P30CA016672) and the funding from noncoding RNA center and used the Functional Proteomics RPPA Core Facility

MicroRNA 603 acts as a tumor suppressor and inhibits triple-negative breast cancer tumorigenesis by targeting elongation factor 2 kinase

Triple negative breast cancer (TNBC) is an aggressive type of breast cancer characterized by the absence of defined molecular targets, including estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2) and is associated with high rates of relapse and distant metastasis despite surgery and adjuvant chemotherapy. The lack of effective targeted therapies for TNBC represents an unmet therapeutic challenge. Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical calcium/calmodulin-dependent serine/threonine kinase that promotes TNBC tumorigenesis, progression, and drug resistance, representing a potential novel molecular target. However, the mechanisms regulating eEF2K expression are unknown. Here, we report that eEF2K protein expression is highly up-regulated in TNBC cells and patient tumors and it is associated with poor patient survival and clinical outcome. We found that loss/reduced expression of miR-603 leads to eEF2K overexpression in TNBC cell lines. Its expression results in inhibition of eEF2K by directly targeting the 3-UTR and the inhibition of tumor cell growth, migration and invasion in TNBC. In vivo therapeutic gene delivery of miR-603 into TNBC xenograft mouse models by systemic administration of miR-603-nanoparticles led to a significant inhibition of eEF2K expression and tumor growth, which was associated with decreased activity of the downstream targets of eEF2K, including Src, Akt, cyclin D1 and c-myc. Our findings suggest that miR-603 functions as a tumor suppressor and loss of miR-603 expression leads to increase in eEF2K expression and contributes to the growth, invasion, and progression of TNBC. Taken together, our data suggest that miR-603-based gene therapy is a potential strategy against TNBC

Exosomal miR-940 maintains SRC-mediated oncogenic activity in cancer cells: a possible role for exosomal disposal of tumor suppressor miRNAs

Exosomes have emerged as important mediators of diverse biological functions including tumor suppression, tumor progression, invasion, immune escape and cell-to-cell communication, through the release of molecules such as mRNAs, miRNAs, and proteins. Here, we identified differentially expressed exosomal miRNAs between normal epithelial ovarian cell line and both resistant and sensitive ovarian cancer (OC) cell lines. We found miR-940 as abundant in exosomes from SKOV3-IP1, HeyA8, and HeyA8-MDR cells. The high expression of miR-940 is associated with better survival in patients with ovarian serous cystadenocarcinoma. Ectopic expression of miR-940 inhibited proliferation, colony formation, invasion, and migration and triggered G0/G1 cell cycle arrest and apoptosis in OC cells. Overexpression of miR-940 also inhibited tumor cell growth in vivo. We showed that proto-oncogene tyrosine-protein kinase (SRC) is directly targeted by miR-940 and that miR-940 inhibited SRC expression at mRNA and protein levels. Following this inhibition, the expression of proteins downstream of SRC, such as FAK, paxillin and Akt was also reduced. Collectively, our results suggest that OC cells secrete the tumor-suppressive miR-940 into the extracellular environment via exosomes, to maintain their invasiveness and tumorigenic phenotype

PRKAR1B-AS2 Long Noncoding RNA Promotes Tumorigenesis, Survival, and Chemoresistance via the PI3K/AKT/mTOR Pathway

Many long noncoding RNAs have been implicated in tumorigenesis and chemoresistance; however, the underlying mechanisms are not well understood. We investigated the role of PRKAR1B-AS2 long noncoding RNA in ovarian cancer (OC) and chemoresistance and identified potential downstream molecular circuitry underlying its action. Analysis of The Cancer Genome Atlas OC dataset, in vitro experiments, proteomic analysis, and a xenograft OC mouse model were implemented. Our findings indicated that overexpression of PRKAR1B-AS2 is negatively correlated with overall survival in OC patients. Furthermore, PRKAR1B-AS2 knockdown-attenuated proliferation, migration, and invasion of OC cells and ameliorated cisplatin and alpelisib resistance in vitro. In proteomic analysis, silencing PRKAR1B-AS2 markedly inhibited protein expression of PI3K-110α and abrogated the phosphorylation of PDK1, AKT, and mTOR, with no significant effect on PTEN. The RNA immunoprecipitation detected a physical interaction between PRKAR1B-AS2 and PI3K-110α. Moreover, PRKAR1B-AS2 knockdown by systemic administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with PRKAR1B-AS2–specific small interfering RNA enhanced cisplatin sensitivity in a xenograft OC mouse model. In conclusion, PRKAR1B-AS2 promotes tumor growth and confers chemoresistance by modulating the PI3K/AKT/mTOR pathway. Thus, targeting PRKAR1B-AS2 may represent a novel therapeutic approach for the treatment of OC patients

Evaluation of nutritional status in pediatric intensive care unit patients: the results of a multicenter, prospective study in Turkey

IntroductionMalnutrition is defined as a pathological condition arising from deficient or imbalanced intake of nutritional elements. Factors such as increasing metabolic demands during the disease course in the hospitalized patients and inadequate calorie intake increase the risk of malnutrition. The aim of the present study is to evaluate nutritional status of patients admitted to pediatric intensive care units (PICU) in Turkey, examine the effect of nutrition on the treatment process and draw attention to the need for regulating nutritional support of patients while continuing existing therapies.Material and MethodIn this prospective multicenter study, the data was collected over a period of one month from PICUs participating in the PICU Nutrition Study Group in Turkey. Anthropometric data of the patients, calorie intake, 90-day mortality, need for mechanical ventilation, length of hospital stay and length of stay in intensive care unit were recorded and the relationship between these parameters was examined.ResultsOf the 614 patients included in the study, malnutrition was detected in 45.4% of the patients. Enteral feeding was initiated in 40.6% (n = 249) of the patients at day one upon admission to the intensive care unit. In the first 48 h, 86.82% (n = 533) of the patients achieved the target calorie intake, and 81.65% (n = 307) of the 376 patients remaining in the intensive care unit achieved the target calorie intake at the end of one week. The risk of mortality decreased with increasing upper mid-arm circumference and triceps skin fold thickness Z-score (OR = 0.871/0.894; p = 0.027/0.024). The risk of mortality was 2.723 times higher in patients who did not achieve the target calorie intake at first 48 h (p = 0.006) and the risk was 3.829 times higher in patients who did not achieve the target calorie intake at the end of one week (p = 0.001). The risk of mortality decreased with increasing triceps skin fold thickness Z-score (OR = 0.894; p = 0.024).ConclusionTimely and appropriate nutritional support in critically ill patients favorably affects the clinical course. The results of the present study suggest that mortality rate is higher in patients who fail to achieve the target calorie intake at first 48 h and day seven of admission to the intensive care unit. The risk of mortality decreases with increasing triceps skin fold thickness Z-score

Etkileşimli ve Geleneksel Kitap Okuma Yöntemlerinin 6 Yaş Grubu Çocukların Öyküleme ve Resimleme Becerilerine Etkisi

The aim of this study was to determine the effect of dialogic and traditional reading methods on the storytelling and drawing skills of six-year-old preschool children. Dialogic and traditional reading methods were applied to two different groups and the application stage occurred over a 12-week period, where 24 picture books were read. Before and after the application period, children's stories and drawings were analyzed. It was revealed in the findings of this study that a statistically significant difference occurred in the storytelling and drawing skills of children in the experimental group (EG) who were read books through the dialogic reading method while no statistically significant difference was determined for the storytelling and drawing skills of the control group (CG) who were read books through the traditional reading method. It is seen that the use of dialogic reading with preschool children leaded to the development of their storytelling and drawing skills and it is predictable that it will ultimately support the long-term development of their language, cognitive and social-emotional skills.Bu araştırmanın amacı, etkileşimli ve geleneksel kitap okuma yöntemlerinin altı yaşındaki okul öncesi dönem çocuklarının öyküleme ve resimleme becerilerine etkisini belirlemektir. İki farklı gruba etkileşimli ve geleneksel kitap okuma yöntemleri kullanılarak 12 hafta boyunca 24 resimli öykü kitabı okunmuştur. Uygulamalardan önce ve sonra çocukların oluşturduğu öyküler ve resimler incelenmiştir. Araştırmanın bulgularında, etkileşimli okuma yöntemi ile kitap okunan deney grubundaki (DG) çocukların öyküleme ve resimleme becerilerinde istatistiksel olarak anlamlı bir farklılık ortaya çıkarken, geleneksel okuma yöntemiyle kitap okunan kontrol grubunun (KG) öyküleme ve resimleme becerilerinde istatistiksel olarak anlamlı bir fark tespit edilememiştir. Okul öncesi dönemdeki çocuklarla etkileşimli okuma yöntemini kullanarak kitap okumanın, çocukların öyküleme ve resimleme becerilerinin gelişimine yol açtığı görülmekte ve nihayetinde uygulamanın dil, bilişsel ve sosyal-duygusal becerilerinin uzun vadeli gelişimini destekleyeceği tahmin edilmektedir

Extraction of L-Aspartic Acid with Reverse Micelle System

The aim of this study is to investigate the extraction L-aspartic acid which is a hydrophobic amino acid with reverse micelle system. Production of amino acids by fermentation has been more important in recent years. These amino acids are obtained in dilute aqueous solutions and have to be separated from excess substrate, inorganic salts and by-products. Recently, separation of amino acids from fermentation media by reverse micelle extraction has received a great deal of attention. In this study, reverse micelle phase includes aliquat-336 as a surfactant, 1-decanol as a co-surfactant and isooctane as an apolar solvent. Experiments were performed at 150 rpm stirring rate, at 30 oC, for 30 min extraction time with equal volumes of reverse micelle and aqueous phases. Concentration of L-aspartic acid was analyzed by liquid chromatography (HPLC). The extraction yield increased with increasing pH and aliquat-336 concentration and with decreasing initial amino acid concentration. Maximum ekstraction yield (68 %) was obtained at pH of 12, surfactant concentration of 200 mM and an initial amino acid concentration of 5 mM

Ters Misel Sistemi ile L-Aspartik Asit Ekstraksiyonu

Bu çalışmanın amacı, hidrofilik bir amino asit olan L-aspartik asitin ekstraksiyonunu ters misel sistemiyle incelemektir. Son yıllarda, amino asitlerin fermentasyonla üretimi giderek önem kazanmaktadır. Bu amino asitler, sulu çözeltilerinde seyreltik olarak bulunurlar ve üretim ortamlarında fazla bulunan substrat, inorganik tuzlar ve yan ürünlerden ayrılması gerekmektedir. Günümüzde ters misel ekstraksiyonu ile amino asitlerin, üretim ortamından ayrılması üzerine çalışmalar yoğunlaşmıştır. Bu çalışmada, ters misel fazı; yüzey aktif madde olarak, aliquat-336, eş yüzey aktif madde olarak, 1-dekanol ve apolar çözücü olarak izooktan içermektedir. Deneyler 150 rpm karıştırma hızında, 30 oC'da, 60 dakika süre ile eşit hacimde ters misel ve sulu fazlar ile gerçekleştirilmiştir. L-aspartik asit derişimi sıvı kromatografi (HPLC) ile analizlenmiştir. Ekstraksiyon verimi, pH ve aliquat-336 derişiminin artmasıyla ve başlangıç amino asit derişiminin azalmasıyla artmıştır. Maksimum ekstraksiyon verimi (% 68) pH 12'de, yüzey aktif madde derişimi 200 mM'da ve başlangıç amino asit derişimi 5 mM'da elde edilmiştir

Optimization of the asymmetric synthesis of (S)-1-phenylethanol using Ispir bean as whole-cell biocatalyst

In this study, enantiomerically pure (S)-1-phenylethanol was produced via asymmetric bioreduction of acetophenone. Ispir bean (Phaseolus vulgaris) was used as an alcohol dehydrogenase (ADH) source since whole cells are cheaper than isolated enzymes. Acetone powder methodology was applied for biocatalyst. Glucose was used as a cosubstrate in-order to regenerate cofactor (NADPH). The reactions were carried out in an orbital shaker whose temperature and agitation rate can be controlled. (S)-1-phenylethanol concentration was analyzed by HPLC using a Chiralcel OB column. Effects of the reaction time, substrate concentration, cosubstrate concentration and biocatalyst concentration on the (S)-1-phenylethanol production were investigated using Response Surface Methodology (RSM). 36 h bioreduction time, 6 mM acetophenone concentration, 25.15 mM glucose concentration, and 175 mg/mL biocatalyst concentration were determined as optimum values. In these conditions, 2.4 mM (S)-1-phenylethanol was obtained in phosphate buffer (pH=7.0) at 30°C with >99% enantiomeric excess