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Background: MiRNAs are non-coding RNA molecules and its function is the regulation of gene expression. In cancer, the deregulation of miRNAs allows them to act as oncogenes or tumor suppressors. From an analysis of the expression of miRNAs in breast cancer (BC) in The Cancer Genome Atlas (TCGA), it was identified that miR-1307-3p is significantly overexpressed in the tumor tissue compared to healthy tissue from patients. So far, in BC, it has only been reported that this miRNA inhibits SMYD4 and that it is involved in resistance to cisplatin through its effect on Mdm4. In this project we propose to identify the role of miR-1307-3p in proliferation, migration, invasion, angiogenesis, and possible targets involved in these processes in BC cells. Methods: RT-qPCR was used to evaluate basal levels of miR-1307-3p in the BC cell lines MDA-MB-231 and MCF-7, and the human epithelial breast MCF-10A cells. Later, we determined the effect of miR-1307-3p on proliferation, migration, and invasion in MDA-MB-231 and MCF-7, and angiogenesis in the HUVEC endothelial cells. All assays were carried out using the miR-1307-3p inhibitor. Finally, nine miRNA-target prediction databases were analyzed to identify potential miR-1307-3p target genes, and their expression was analyzed by RT-qPCR in a designed 384-well plate. Results: We found that miR-1307-3p is overexpressed in MDA-MB-231 and MCF-7, compared to MCF-10A cells. We also identified that transfection with the miR-1307-3p inhibitor causes a significant decrease in the processes of proliferation, migration, invasion, and angiogenesis, when compared with untreated or negative control transfected cells. For its part, prediction databases analysis allowed us to identify 19 potential targets of miR-1307-3p. Finally, by RT-qPCR, the overexpression of 3 and the downregulation of 2 genes were confirmed. Conclusions: MiR-1307-3p is overexpressed in BC cells. Furthermore, miR-1307-3p induces the processes of proliferation, migration and invasion in BC cells, and angiogenesis in HUVEC cells. These observations suggest that miR-1307-3p can acts as an onco-miRNA. In addition, the expression of 5 of the predicted target genes were altered by miR-1307-3p inhibitor. Further analysis to validate the implication of this miR-1307-3p targets are needed to understand its importance in BC. Citation Format: José Roberto Estupiñan Jimenez, Valeria Villarreal-García, Ricardo Noriega, Recep Bayraktar, Diana Reséndez-Pérez, Cristina Rodríguez-Padilla, José Manuel Vázquez-Guillén, Fermín Mar-Aguilar, Gabriel Lopez-Berestein, Pablo E. Vivas-Mejía, Vianey Gonzalez-Villasana. Functional effect of miR-1307-3p on breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-04.

Background: Breast cancer (BC) is the most diagnosed cancer in women worldwide. MicroRNAs (miRNAs) are involved in different processes of BC and their deregulation can cause them to act as oncogenes or tumor suppressors, participating in cancer progression or also as therapeutic target. Using The Cancer Genome Atlas (TCGA) database, we found that miR-660-5p is significantly overexpressed and associated with poor survival in patients with this pathology. Moreover, it is reported that miR-660-5p can induce BC progression through transcription factor CP2 (TFCP2) and the down regulation of tet-eleven translocation 2 (TET2). In this project, we propose to identify the role of miR-660-5p in proliferation, migration, invasion, angiogenesis, and possible targets involved in these processes in BC cell lines. Methods: Basal levels of miR-660-5p were determined in BC cells MDA-MB-231 and MCF-7, and in human epithelial breast cells MCF-10A by RT-qPCR. The effect of miR-660-5p was evaluated on proliferation, migration, and invasion processes using MDA-MB-231 and MCF-7 cells. HUVEC cells were used to assess angiogenesis. All cell lines were transfected with miR-660-5p inhibitor. Analysis of nine miRNA-target prediction databases was made to identify targets of miR-660-5p. We selected the target genes predicted by at least three of these programs, and their expression was evaluated by RT-qPCR in a customized 384-well plate. Results: We found that miR-660-5p is significantly upregulated in MDA-MB-231 and MCF-7, compared to MCF-10A cells. In addition, we observed a significant decrease in proliferation, migration, and invasion of BC cells transfected with miR-660-5p inhibitor, compared to non-treated cells and miRNA inhibitor negative control treated cells. Similarly, we observed a significant decrease in angiogenesis of HUVEC cells transfected with miR-660-5p inhibitor. Furthermore, of all the miR-660-5p targets identified by prediction databases 21 were selected, and of these, 7 were observed upregulated and 1 downregulated. Conclusions: The results show that miR-660-5p is upregulated and involved in proliferation, migration, invasion, and angiogenesis of BC, which may lead us to suggest that this miRNA act as an onco-miRNA. In addition, seven potential miR-660-5p target genes were identified, but further validation assays are needed to clarify their implication in this disease. Citation Format: Valeria Villarreal-García, José Roberto Estupiñan Jimenez, Ricardo Noriega, Recep Bayraktar, Diana Reséndez-Pérez, Cristina Rodríguez-Padilla, José Manuel Vázquez-Guillén, Gabriel Lopez-Berestein, Pablo E. Vivas-Mejía, Vianey Gonzalez-Villasana. Effect of miR-660-5p in breast cancer progression [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-22-03.

In the new media where there are many presentations and representations, the audience who are increasingly fragmented, are integrated around different thematic areas, and more specific interests that affect their mass characteristics. In this study, ASMR the phenomenon, which is among the popular contents of YouTube, and has begun to gain a place in the mainstream media, is discussed as a new contextual category that corresponds to the more specific interests of the audience and within the scope of narrowcasting. The expansions that ASMR brings to both narrowcasting and fragmented audience are presented. In this context, ASMR videos were observed by the netnography method during a year on YouTube, 8 Turkish ASMR content creators/ASMRtists were interviewed with, and the audience comments in the three most watched videos of these ASMRtists on YouTube were examined. Among the more significant findings of the study are that the ASMR has 14 content categories in the context of narrowcasting, that the audience who expect relaxation/therapy with ASMR make requests like an active content producer/prosumer, and they need media mediation even so as to fall asleep.

Blood biochemistry of mice treated with liposomal miR-34a or control mimic.

Supplementary Data includes Supplementary Figures 1-6 and Supplementary Tables 1-3. Supplementary Figure 1 shows microscopic images for the characterization of exosomes. Supplementary Figure 2 shows the qPCR results of miR-6126 overexpression in miR-6126 mimic treated ovarian cancer cells (HeyA8, HeyA8-MDR, Skkov3-ip1, SKOV3-TR) Supplementary Figure 3 shows western blotting and qPCR results that shows the ingibition of Integrin beta-1 expression at protein and mRNA level upon miR-6126 mimic treatment. Supplementary Figure 4 shows the size distribution graphs of exosomes isolated from CTL mimic, miR-6126 mimic , CTL inhibitor, miR-6126 inhibitor treated SKOV3-IP1 cells. Supplementary Figure 5 shows ITGB1 mRNA levels in different ovarian cancer exosomes. Supplementary Figure 6 shows in vivo down regulation of ITGB1. Supplementary Table 1 shows demographic information of ovarian cancer patients whose tissue samples used in quantification of miR-6126 expression. Supplementary Table 2 shows raw microarray data for miR-6126 Supplementary Table 3 shows z-score values for miR-6126 for each exosome and cell sample.

In vivo nanoliposomal miR-34a treatment is safe and effectively suppresses eEF2K and FOXM1 protein expression in TNBC tumors.

Purpose: Recent studies indicated that dysregulation of noncoding RNAs (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 miR-34a in FOXM1/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 nanoparticles 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. Clin Cancer Res; 24(17); 4225–41. ©2018 AACR.

Figure Legends for Supplementary Figures

Supplementary Methods includes experimental details on western blotting, invasion, wound healing, tube formation, EdU cell proliferation assays, quantitativce RT-PCR, luciferase reporter assay, gene expression analysis and Ingenuity Pathway Analysis.

Background: Ultraconserved regions or elements (UCEs) are greater than 100-base pairs in length and are perfectly (100%) conserved across large evolutionary distances in the genomes of at least 3 of 5 placental mammals: human, cow, dog, rat, and mouse. Most UCEs are located in non-coding regions of the genome; however, some UCEs overlap with coding exons. In addition, some UCEs have been found to be transcriptionally active as long non-coding RNAs and are involved in a variety of cellular processes such as cellular proliferation. Although a few UCE sequence variations were reported to be associated with human diseases, the molecular functions of somatic mutations in UCEs remain largely unexplained in human cancers. In this study, we characterized the distribution of somatic UCE mutations throughout a spectrum of cancers and investigated the biological functions of cancer associated UCEs. Methods: We examined somatic UCE mutations in 2,449 cases of 22 cancer types using the PCAWG and ICGC platforms. To validate these mutation patterns, UCE sequencing was performed on the Illumina NovaSeq 6000 platform for MD Anderson patient cohorts. A custom AsCpf1 guide library was built to identify potential regulatory UCE functions in colorectal cancer models, and 3 guides were designed to target 2,247 UCEs. The proliferation ratio of knockout UCEs was analyzed. We then generated stable mutated UCE clones using the CRISPR AsCpf1 technology in colorectal cancer cells. We conducted RNA sequencing (RNA-seq) of mutated UCE cells. The expression of target proteins and genes were analyzed by qRT-PCR and western blotting. To address the effects of the mutated UCE_11311 tumor growth, DLD1 cells were injected in athymic nude mice. Results: We analyzed the WGS data on 2,449 cases of 22 cancer types and identified 24,039 somatic mutations in 10,090 (73.46%) UCEs. These were mostly located in non-coding DNAs, mainly in introns.Based on the RNA-seq data, one of the strongest impacts of mutated UCEs was on ARID1B. We confirmed that specific mutated UCE decreased ARID1B mRNA and protein levels. Our in vivo results demonstrated that UCE mutations enhanced the tumorigenicity of DLD1 xenograft tumors. As a summary, UCE_11311 has a transcriptional enhancer activity on the ARID1B gene and UCE_11311 mutations are actively participating in the tumorigenesis at least in part through regulating ARID1B expression. Discussion: We identified hundreds of unexplored tumorigenic UCEs that need to be further characterized functionally and clinically, and proved one of them to be a transcriptional enhancer of the tumor suppressor ARID1B. Collectively, these data support the concept that certain somatic UCE mutations are frequent and functional in cancer evolution, acting as driver mutations that can be used as new therapeutic targets; other somatic UCE mutations are cancer specific or patient specific, are markers of aggressiveness, and can be used to personalized therapy. Citation Format: Recep Bayraktar, Yitao Tang, Mihnea P. Dragomir, Linda Fabris, Giulio F. Draetta, Han Liang, George A. Calin. The mutational landscape of ultraconserved elements in human cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5750.

Deregulation of noncoding RNAs, including microRNAs (miRs), is implicated in the pathogenesis of many human cancers, including breast cancer. Through extensive analysis of The Cancer Genome Atlas, we found that expression of miR-22-3p is markedly lower in triple-negative breast cancer (TNBC) than in normal breast tissue. The restoration of miR-22-3p expression led to significant inhibition of TNBC cell proliferation, colony formation, migration, and invasion. We demonstrated that miR-22-3p reduces eukaryotic elongation factor 2 kinase (eEF2K) expression by directly binding to the 3′ untranslated region of eEF2K mRNA. Inhibition of EF2K expression recapitulated the effects of miR-22-3p on TNBC cell proliferation, motility, invasion, and suppression of phosphatidylinositol 3-kinase/Akt and Src signaling. Systemic administration of miR-22-3p in single-lipid nanoparticles significantly suppressed tumor growth in orthotopic MDA-MB-231 and MDA-MB-436 TNBC models. Evaluation of the tumor response, following miR-22-3p therapy in these models using a novel mathematical model factoring in various in vivo parameters, demonstrated that the therapy is highly effective against TNBC. These findings suggest that miR-22-3p functions as a tumor suppressor by targeting clinically significant oncogenic pathways and that miR-22-3p loss contributes to TNBC growth and progression. The restoration of miR-22-3p expression is a potential novel noncoding RNA-based therapy for TNBC., Graphical Abstract, Our study demonstrates that microRNA-22-3p (miR-22-3p) acts as a tumor suppressor in which its expression is markedly reduced in TNBC tumors. Restoration of its expression by miR-22-3p nanotherapy suppress tumor growth and progression by directly inhibiting of eEF2K-PI3K/AKT and Src signaling. miR-22-3p-based RNA therapy may be novel treatment for TNBC.

The primary objective of the present study is to evaluate the effect of single-layer locked versus single-layer unlocked uterine closure techniques on the development of uterine niche. The secondary objective is to evaluate the effect of the ratio of lower uterine segment (LUS) to upper uterine segment (UUS) on the development of uterine niche, which was not previously investigated in literature but which the authors consider to be a major risk factor for the development of uterine niche.Included in this randomized study were 194 patients who were admitted to the Department of Obstetrics and Gynecology at Health Sciences University Antalya Training and Research Hospital and who underwent cesarean section (CS) due to any reason between October 2017 and May 2018. Two different techniques were used in the closure of hysterotomy: Single-layer locked continuous suturing (Group 1) and single-layer unlocked continuous suturing (Group 2). During surgery, the thicknesses of the LUS and UUS were measured using a sterile scale prior to hysterotomy closure. The patients were evaluated for the development of uterine niche at postoperative six months by transvaginal ultrasound.Control transvaginal ultrasound performed at six months after surgery revealed uterine niches in 58 out of 194 patients (29.29%; 34 patients in Group 1 [34.3%] and 24 patients in Group 2 [25.3%]). No significant difference was noted in terms of the development of uterine niche between the two groups (The present study found no statistically significant difference in niche size between the two groups. However, the study reports that the ratio of upper to lower uterine segment that was not previously investigated in literature is a major risk factor for the development of uterine niche.

The human genome contains 481 ultraconserved regions (UCRs), which are genomic stretches of over 200 base pairs conserved among human, rat, and mouse. The majority of these regions are transcriptionally active (T-UCRs), and several have been found to be differentially expressed in tumours. Some T-UCRs have been functionally characterized, but of those few have been associated to breast cancer (BC). Using TCGA data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with oestrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. The expression levels of 12 T-UCRs were further analysed in a cohort of 82 Brazilian BC patients using RT-qPCR. We found that uc.147 is high expressed in luminal A and B patients. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is located in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic trancript, and its sequence was confirmed by RACE. The silencing of uc.147 increases apoptosis, arrests cell cycle, and reduces cell viability and colony formation in BC cell lines. Additionally, we identifed 19 proteins that interact with lnc-uc.147 through mass spectrometry and demonstrated a high correlation of lnc-uc.147 with the neighbour gene expression and miR-18 and miR-190b. This is the first study to analyse the expression of all T-UCRs in BC and to functionally assess the lnc-uc.147.

This is an Accepted Manuscript of an article published by Taylor & Francis in RNA Biology on 13. august 2021, available (including supplementary) material online: https://doi.org/10.1080/15476286.2021.1952757. The human genome contains 481 ultraconserved regions (UCRs), which are genomic stretches of over 200 base pairs conserved among human, rat, and mouse. The majority of these regions are transcriptionally active (T-UCRs), and several have been found to be differentially expressed in tumours. Some T-UCRs have been functionally characterized, but of those few have been associated to breast cancer (BC). Using TCGA data, we found 302 T-UCRs related to clinical features in BC: 43% were associated with molecular subtypes, 36% with oestrogen-receptor positivity, 17% with HER2 expression, 12% with stage, and 10% with overall survival. The expression levels of 12 T-UCRs were further analysed in a cohort of 82 Brazilian BC patients using RT-qPCR. We found that uc.147 is high expressed in luminal A and B patients. For luminal A, a subtype usually associated with better prognosis, high uc.147 expression was associated with a poor prognosis and suggested as an independent prognostic factor. The lncRNA from uc.147 (lnc-uc.147) is located in the nucleus. Northern blotting results show that uc.147 is a 2,8 kb monoexonic trancript, and its sequence was confirmed by RACE. The silencing of uc.147 increases apoptosis, arrests cell cycle, and reduces cell viability and colony formation in BC cell lines. Additionally, we identifed 19 proteins that interact with lnc-uc.147 through mass spectrometry and demonstrated a high correlation of lnc-uc.147 with the neighbour gene expression and miR-18 and miR-190b. This is the first study to analyse the expression of all T-UCRs in BC and to functionally assess the lnc-uc.147.

Background Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood. Methods To investigate the role of exosomal miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments. Findings Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFβ receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages. Interpretation Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.

KRAS is one of the most frequently mutated proto-oncogenes in pancreatic ductal adenocarcinoma (PDAC) and aberrantly activated in triple-negative breast cancer (TNBC). A profound role of microRNAs (miRNAs) in the pathogenesis of human cancer is being uncovered, including in cancer therapy. Using in silico prediction algorithms, we identified miR-873 as a potential regulator of KRAS, and we investigated its role in PDAC and TNBC. We found that reduced miR-873 expression is associated with shorter patient survival in both cancers. miR-873 expression is significantly repressed in PDAC and TNBC cell lines and inversely correlated with KRAS levels. We demonstrate that miR-873 directly bound to the 3′ UTR of KRAS mRNA and suppressed its expression. Notably, restoring miR-873 expression induced apoptosis; recapitulated the effects of KRAS inhibition on cell proliferation, colony formation, and invasion; and suppressed the activity of ERK and PI3K/AKT, while overexpression of KRAS rescued the effects mediated by miR-873. Moreover, in vivo delivery of miR-873 nanoparticles inhibited KRAS expression and tumor growth in PDAC and TNBC tumor models. In conclusion, we provide the first evidence that miR-873 acts as a tumor suppressor by targeting KRAS and that miR-873-based gene therapy may be a therapeutic strategy in PDAC and TNBC. Keywords: KRAS, oncogene, non-coding RNA, microRNA, ncRNA, miR-873, proliferation, invasion, gene regulation, tumorigenesis, gene silencing, therapy, nanoparticles, pancreatic cancer, liposomes, breast cancer, triple-negative breast cancer

Richter syndrome is the name given to the transformation of the most frequent type of leukemia, chronic lymphocytic leukemia, into an aggressive lymphoma. Patients with Richter syndrome have limited response to therapies and dismal survival. The underlying mechanisms of transformation are insufficiently understood and there is a major lack of knowledge regarding the roles of microRNA that have already proven to be causative for most cases of chronic lymphocytic leukemia. Here, by using four types of genomic platforms and independent sets of patients from three institutions, we identified microRNA involved in the transformation of chronic lymphocytic leukemia to Richter syndrome. The expression signature is composed of miR-21, miR-150, miR-146b and miR-181b, with confirmed targets significantly enriched in pathways involved in cancer, immunity and inflammation. In addition, we demonstrated that genomic alterations may account for microRNA deregulation in a subset of cases of Richter syndrome. Furthermore, network analysis showed that Richter transformation leads to a complete rearrangement, resulting in a highly connected microRNA network. Functionally, ectopic overexpression of miR-21 increased proliferation of malignant B cells in multiple assays, while miR-150 and miR-26a were downregulated in a chronic lymphocytic leukemia xenogeneic mouse transplantation model. Together, our results suggest that Richter transformation is associated with significant expression and genomic loci alterations of microRNA involved in both malignancy and immunity.

Medya ekosistemini anlamaya yönelik çabalara katkı sağlayan teknolojik determinizm medyadaki gelişmeleri nedensellik ilişkisi üzerinden açıklamaya çalışmaktadır. Bu nedensellik olgusu kendisinden önceki medyumu değişime uğratan yeni araçların genel hareket noktalarını saptamaya yönelik ilkeler ortaya koymaktadır. Bu doğrultuda geliştirilen iyileştirme kuramı medya ekosistemini anlamlandırmak için kapsamlı bir çerçeve sunar. Bu çalışmada, televizyon yayıncılığının iletim biçimleri olan karasal yayıncılık, kablolu yayıncılık, uydu yayıncılığı ve internet yayıncılığı ile ortaya çıkan dönüşümler hem televizyon yayıncılığı hem de izleyici tercihleri açısından literatüre dayalı olarak teorik bir çerçeveden ele alınmaya çalışılmıştır. Bu kapsamda, televizyon yayıncılığının ve izleyici tercihlerinin günümüzdeki durumunu betimleyici bir yaklaşımla ortaya koyabilmek için iyileştirme kuramı çerçevesinde ilgili literatür taranmıştır. Literatürde, televizyon yayıncılığı alanında meydana gelen iyileştirmeler neticesinde izleyici tercihlerinde çeşitliliğin arttığı, izleyicilerin özel ilgilerine yönelik bir yayıncılık türünün geliştiği ve etkileşimli uygulamalar ile izleyicilerin daha dinamik bir izleme süreci içerisine girdikleri tespit edilmiştir.

Circulating miRNAs are known to play important roles in intercellular communication. However, the effects of exosomal miRNAs on cells are not fully understood.To investigate the role of exosomal miR-1246 in ovarian cancer (OC) microenvironment, we performed RPPA as well as many other in vitro functional assays in ovarian cancer cells (sensitive; HeyA8, Skov3ip1, A2780 and chemoresistant; HeyA8-MDR, Skov3-TR, A2780-CP20). Therapeutic effect of miR-1246 inhibitor treatment was tested in OC animal model. We showed the effect of OC exosomal miR-1246 uptake on macrophages by co-culture experiments.Substantial expression of oncogenic miR-1246 OC exosomes was found. We showed that Cav1 gene, which is the direct target of miR-1246, is involved in the process of exosomal transfer. A significantly worse overall prognosis were found for OC patients with high miR-1246 and low Cav1 expression based on TCGA data. miR-1246 expression were significantly higher in paclitaxel-resistant OC exosomes than in their sensitive counterparts. Overexpression of Cav1 and anti-miR-1246 treatment significantly sensitized OC cells to paclitaxel. We showed that Cav1 and multi drug resistance (MDR) gene is involved in the process of exosomal transfer. Our proteomic approach also revealed that miR-1246 inhibits Cav1 and acts through PDGFβ receptor at the recipient cells to inhibit cell proliferation. miR-1246 inhibitor treatment in combination with chemotherapy led to reduced tumor burden in vivo. Finally, we demonstrated that when OC cells are co-cultured with macrophages, they are capable of transferring their oncogenic miR-1246 to M2-type macrophages, but not M0-type macrophages.Our results suggest that cancer exosomes may contribute to oncogenesis by manipulating neighboring infiltrating immune cells. This study provide a new mechanistic therapeutic approach to overcome chemoresistance and tumor progression through exosomal miR-1246 in OC patients.

Myelofibrosis (MF) is a myeloproliferative neoplasm characterized by cytopenia and extramedullary hematopoiesis, resulting in splenomegaly. Multiple pathological mechanisms (e.g., circulating cytokines and genetic alterations, such as JAKV617F mutation) have been implicated in the etiology of MF, but the molecular mechanism causing resistance to JAK2V617F inhibitor therapy remains unknown. Among MF patients who were treated with the JAK inhibitor ruxolitinib, we compared noncoding RNA profiles of ruxolitinib therapy responders versus nonresponders and found miR-543 was significantly upregulated in nonresponders. We validated these findings by reverse transcription–quantitative PCR. in this same cohort, in 2 additional independent MF patient cohorts from the United States and Romania, and in a JAK2V617F mouse model of MF. Both in vitro and in vivo models were used to determine the underlying molecular mechanism of miR-543 in MF. Here, we demonstrate that miR-543 targets the dioxygenases ten-eleven translocation 1 (TET1) and 2 (TET2) in patients and in vitro, causing increased levels of global 5-methylcytosine, while decreasing the acetylation of histone 3, STAT3, and tumor protein p53. Mechanistically, we found that activation of STAT3 by JAKs epigenetically controls miR-543 expression via binding the promoter region of miR-543. Furthermore, miR-543 upregulation promotes the expression of genes related to drug metabolism, including CYP3A4, which is involved in ruxolitinib metabolism. Our findings suggest miR-543 as a potentially novel biomarker for the prognosis of MF patients with a high risk of treatment resistance and as a potentially new target for the development of new treatment options The work in GAC’s laboratory was partly supported by NIH/National Center for Advancing Translational Sciences grant UH3TR00943-01, the NIH/National Cancer Institute (NCI) grant 1 R01 CA182905-01, U54 grant UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, Team Department of Defense grant CA160445P1, a Ladies Leukemia League grant, a Chronic Lymphocytic Leukemia Moonshot Flagship project, a Sister Institution Network Fund 2017 grant, and the Estate of C. G. Johnson, Jr. EFM was supported in part by award number P50 CA140388 from the NCI and by the NIH Clinical Research Loan Repayment Program. AMS was supported by Fundação para a Ciência e a Tecnologia through fellowship SFRH/BD/85968/2012. ZJZ’s work was supported by grants from the MPN Foundation and the Oklahoma Center for the Advancement of Science and Technology. DG, MPD, and IBN were supported in part by a Programul Opera¿ional Competitivitate grant nr35/01.09.2016, ID 37_796, titled “Clinical and economical impact of personalized targeted anti-mi-croRNA therapies in reconverting lung cancer chemoresistance” (CANTEMIR). NSDA was supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, BEPE 2016/09349-4.

ObjectiveTo investigate the function of a novel primate-specific long non-coding RNA (lncRNA), named FLANC, based on its genomic location (co-localised with a pyknon motif), and to characterise its potential as a biomarker and therapeutic target.DesignFLANC expression was analysed in 349 tumours from four cohorts and correlated to clinical data. In a series of multiple in vitro and in vivo models and molecular analyses, we characterised the fundamental biological roles of this lncRNA. We further explored the therapeutic potential of targeting FLANC in a mouse model of colorectal cancer (CRC) metastases.ResultsFLANC, a primate-specific lncRNA feebly expressed in normal colon cells, was significantly upregulated in cancer cells compared with normal colon samples in two independent cohorts. High levels of FLANC were associated with poor survival in two additional independent CRC patient cohorts. Both in vitro and in vivo experiments demonstrated that the modulation of FLANC expression influenced cellular growth, apoptosis, migration, angiogenesis and metastases formation ability of CRC cells. In vivo pharmacological targeting of FLANC by administration of 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine nanoparticles loaded with a specific small interfering RNA, induced significant decrease in metastases, without evident tissue toxicity or pro-inflammatory effects. Mechanistically, FLANC upregulated and prolonged the half-life of phosphorylated STAT3, inducing the overexpression of VEGFA, a key regulator of angiogenesis.ConclusionsBased on our findings, we discovered, FLANC as a novel primate-specific lncRNA that is highly upregulated in CRC cells and regulates metastases formation. Targeting primate-specific transcripts such as FLANC may represent a novel and low toxic therapeutic strategy for the treatment of patients.

BACKGROUND & AIMS: Chromosomal instability (CIN) is a carcinogenesis event that promotes metastasis and resistance to therapy by unclear mechanisms. Expression of the colon cancer-associated transcript 2 gene (CCAT2), which encodes a long noncoding RNA (lncRNA), associates with CIN, but little is known about how CCAT2 lncRNA regulates this cancer enabling characteristic.METHODS: We performed cytogenetic analysis of colorectal cancer (CRC) cell lines (HCT116, KM12C/SM, and HT29) overexpressing CCAT2 and colon organoids from C57BL/6N mice with the CCAT2 transgene and without (controls). CRC cells were also analyzed by immunofluorescence microscopy, gamma-H2AX, and senescence assays. CCAT2 transgene and control mice were given azoxymethane and dextran sulfate sodium to induce colon tumors. We performed gene expression array and mass spectrometry to detect downstream targets of CCAT2 lncRNA. We characterized interactions between CCAT2 with downstream proteins using MS2 pull-down, RNA immunoprecipitation, and selective 2'-hydroxyl acylation analyzed by primer extension analyses. Downstream proteins were overexpressed in CRC cells and analyzed for CIN. Gene expression levels were measured in CRC and non-tumor tissues from 5 cohorts, comprising more than 900 patients.RESULTS: High expression of CCAT2 induced CIN in CRC cell lines and increased resistance to 5-fluorouracil and oxaliplatin. Mice that expressed the CCAT2 transgene developed chromosome abnormalities, and colon organoids derived from crypt cells of these mice had a higher percentage of chromosome abnormalities compared with organoids from control mice. The transgenic mice given azoxymethane and dextran sulfate sodium developed more and larger colon polyps than control mice given these agents. Microarray analysis and mass spectrometry indicated that expression of CCAT2 increased expression of genes involved in ribosome biogenesis and protein synthesis. CCAT2 lncRNA interacted directly with and stabilized BOP1 ribosomal biogenesis factor (BOP1). CCAT2 also increased expression of MYC, which activated expression of BOP1. Overexpression of BOP1 in CRC cell lines resulted in chromosomal missegregation errors, and increased colony formation, and invasiveness, whereas BOP1 knockdown reduced viability. BOP1 promoted CIN by increasing the active form of aurora kinase B, which regulates chromosomal segregation. BOP1 was overexpressed in polyp tissues from CCAT2 transgenic mice compared with healthy tissue. CCAT2 lncRNA and BOP1 mRNA or protein were all increased in microsatellite stable tumors (characterized by CIN), but not in tumors with microsatellite instability compared with nontumor tissues. Increased levels of CCAT2 lncRNA and BOP1 mRNA correlated with each other and with shorter survival times of patients.CONCLUSIONS: We found that overexpression of CCAT2 in colon cells promotes CIN and carcinogenesis by stabilizing and inducing expression of BOP1 an activator of aurora kinase B. Strategies to target this pathway might be developed for treatment of patients with microsatellite stable colorectal tumors.

Small non-coding microRNAs (miRNAs) are instrumental in physiological processes, such as proliferation, cell cycle, apoptosis, and differentiation, processes which are often disrupted in diseases like cancer. miR-155 is one of the best conserved and multifunctional miRNAs, which is mainly characterized by overexpression in multiple diseases including malignant tumors. Altered expression of miR-155 is found to be associated with various physiological and pathological processes, including hematopoietic lineage differentiation, immune response, inflammation, and tumorigenesis. Furthermore, miR-155 drives therapy resistance mechanisms in various tumor types. Therefore, miR-155-mediated signaling pathways became a potential target for the molecular treatment of cancer. In this review, we summarize the current findings of miR-155 in hematopoietic lineage differentiation, the immune response, inflammation, and cancer therapy resistance. Furthermore, we discuss the potential of miR-155-based therapeutic approaches for the treatment of cancer.

Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK) has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER), and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.]) administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer.

Mammalian cells can release different types of extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies. Accumulating evidence suggests that EVs play a role in cell‐to‐cell communication within the tumor microenvironment. EVs’ components, such as proteins, noncoding RNAs [microRNAs (miRNAs), and long noncoding RNAs (lncRNAs)], messenger RNAs (mRNAs), DNA, and lipids, can mediate paracrine signaling in the tumor microenvironment. Recently, miRNAs encapsulated in secreted EVs have been identified in the extracellular space. Mature miRNAs that participate in intercellular communication are released from most cells, often within EVs, and disseminate through the extracellular fluid to reach remote target cells, including tumor cells, whose phenotypes they can influence by regulating mRNA and protein expression either as tumor suppressors or as oncogenes, depending on their targets. In this review, we discuss the roles of miRNAs in intercellular communication, the biological function of extracellular miRNAs, and their potential applications for diagnosis and therapeutics. We will give examples of miRNAs that behave as hormones.

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.

Current anti-angiogenic therapy for cancer is based mainly on inhibition of the vascular endothelial growth factor pathway. However, due to the transient and only modest benefit from such therapy, additional approaches are needed. Deregulation of microRNAs (miRNAs) has been demonstrated to be involved in tumor angiogenesis and offers opportunities for a new therapeutic approach. However, effective miRNA-delivery systems are needed for such approaches to be successful. In this study, miRNA profiling of patient data sets, along with in vitro and in vivo experiments, revealed that miR-204-5p could promote angiogenesis in ovarian tumors through THBS1. By binding with scavenger receptor class B type 1 (SCARB1), reconstituted high-density lipoprotein-nanoparticles (rHDL-NPs) were effective in delivering miR-204-5p inhibitor (miR-204-5p-inh) to tumor sites to suppress tumor growth. These results offer a new understanding of miR-204-5p in regulating tumor angiogenesis.

Multiple sclerosis (MS) is an imflammatory disease of central nervous system caused by genetic and environmental factors that remain largely unknown. Autophagy is the process of degradation and recycling of damaged cytoplasmic organelles, macromolecular aggregates, and long-lived proteins. Malfunction of autophagy contributes to the pathogenesis of neurological diseases, and autophagy genes may modulate the T cell survival. We aimed to examine the expression levels of autophagy-related genes. The blood samples of 95 unrelated patients (aged 17-65years, 37 male, 58 female) diagnosed as MS and 95 healthy controls were used to extract the RNA samples. After conversion to single stranded cDNA using polyT priming: the targeted genes were pre-amplified, and 96×78 (samples×primers) qRT-PCR reactions were performed for each primer pair on each sample on a 96.96 array of Fluidigm BioMark™. Compared to age- and sex-matched controls, gene expression levels of ATG16L2, ATG9A, BCL2, FAS, GAA, HGS, PIK3R1, RAB24, RGS19, ULK1, FOXO1, HTT were significantly altered (false discovery rate

Mammalian Sirtuins have been shown to perform distinct cellular functions and deregulated expression of these genes was reported to be involved in the development of various malignancies including breast cancer. An increasing number of evidence indicates that Sirtuins have both tumor promoter and tumor suppressor functions. However, the roles of Sirtuins have not been well-reported in breast cancer. In the present study, quantitative expression levels of Sirtuins (SIRT1-7) in breast cancer patients and breast cancer cell lines (MCF-7 and SKBR3) and control cell line (CRL-4010) were assessed by using a high-throughput real-time PCR method. As a result, Sirtuins were found to be differentially expressed in breast cancer tissues and cancer cell lines. Particularly, expressions of SIRT1 and SIRT4 were found to be significantly down-regulated in breast cancer tissues and SKBR3 breast cancer cells. In contrast, SIRT2, SIRT3, and SIRT5 genes were shown to be up-regulated in our study. Although SIRT6 and SIRT7 were also up-regulated in breast cancer tissues, these expression changes were statistically insignificant. Additionally, SIRT2, SIRT3, SIRT5, SIRT6 and SIRT7 were found to be differentially expressed in breast cancer cell lines. Yet, these changes were not well-correlated with tissue expression levels. In conclusion, Sirtuin family of genes shows differential expressions in breast cancer tissues and cells and SIRT1 and SIRT4 seem to play key tumor suppressor roles in breast cancer development. Herein, we report expression levels of Sirtuin family of genes in both breast cancer tissues and cancer cell lines simultaneously.

MicroRNAs (miRNAs) are small noncoding RNAs that target specific mRNAs through interaction with complementary sequences usually found in the 3'-untranslated regions (UTRs) of target mRNAs. miRNAs have been shown to play a fundamental role in the management of chronic lymphocytic leukemia (CLL) by modulating gene expression patterns and cellular signaling pathways. In recent years, several studies have focused on the role of regulatory miRNAs in the pathogenesis of CLL. Aberrant expression of CLL-specific miRNAs has emerged as therapeutic and diagnostic biomarkers in patients with CLL. Here, we describe a method for the quantification of miRNAs in malignant B cells from the mononuclear cell compartment, isolated from peripheral blood. We focus on the isolation of human blood monocytes by Ficoll-Paque gradient centrifugation, total RNA extraction from human peripheral blood mononuclear cells, and quantitative reverse transcription (qRT)-PCR, which is useful for the measurement of miRNAs in monocytes isolated from blood samples.

MicroRNAs (miRNAs) are small noncoding RNAs that target specific mRNAs through interaction with complementary sequences usually found in the 3'-untranslated regions (UTRs) of target mRNAs. miRNAs have been shown to play a fundamental role in the management of chronic lymphocytic leukemia (CLL) by modulating gene expression patterns and cellular signaling pathways. In recent years, several studies have focused on the role of regulatory miRNAs in the pathogenesis of CLL. Aberrant expression of CLL-specific miRNAs has emerged as therapeutic and diagnostic biomarkers in patients with CLL. Here, we describe a method for the quantification of miRNAs in malignant B cells from the mononuclear cell compartment, isolated from peripheral blood. We focus on the isolation of human blood monocytes by Ficoll-Paque gradient centrifugation, total RNA extraction from human peripheral blood mononuclear cells, and quantitative reverse transcription (qRT)-PCR, which is useful for the measurement of miRNAs in monocytes isolated from blood samples.

miR-145, miR-155, and miR-382 have been proposed as noninvasive biomarkers to distinguish breast cancer patients from healthy individuals. However, it is unknown if these three miRNAs are secreted by exosomes. Thus, we hypothesized that miR-145, miR-155, and miR-382 in breast cancer patients are present in exosomes. We isolated exosomes from serum of breast cancer patients and healthy donors, then we characterized them according to their shape, size, and exosome markers by scanning electron microscopy, atomic force microscopy, nanoparticle tracking analysis (NTA), and Western blot and determined the exosome concentration in all samples by NTA. Later, exosomal small RNA extraction was done to determine the expression levels of miR-145, miR-155, and miR-382 by qRT-PCR. We observed a round shape of exosomes with a mean size of 119.84 nm in breast cancer patients and 115.4 nm in healthy donors. All exosomes present the proteins CD63, Alix, Tsg, CD9, and CD81 commonly used as markers. Moreover, we found a significantly high concentration of exosomes in breast cancer patients with stages I, III, and IV compared to healthy donors. We detected miR-145, miR-155, and miR-382 in the exosomes isolated from serum of breast cancer patients and healthy donors. Our results show that the exosomes isolated from the serum of breast cancer patients and healthy donors contains miR-145, miR-155, and miR-382 but not in a selective manner in breast cancer patients. Moreover, our data support the association between exosome concentration and the presence of breast cancer, opening the possibility to study how miRNAs packaged into exosomes play a role in BC progression.

Current antiangiogenesis therapy relies on inhibiting newly developed immature tumor blood vessels and starving tumor cells. This strategy has shown transient and modest efficacy. Here, we report a better approach to target cancer-associated endothelial cells (ECs), reverse permeability and leakiness of tumor blood vessels, and improve delivery of chemotherapeutic agents to the tumor. First, we identified deregulated microRNAs (miRs) from patient-derived cancer-associated ECs. Silencing these miRs led to decreased vascular permeability and increased maturation of blood vessels. Next, we screened a thioaptamer (TA) library to identify TAs selective for tumor-associated ECs. An annexin A2-targeted TA was identified and used for delivery of miR106b-5p and miR30c-5p inhibitors, resulting in vascular maturation and antitumor effects without inducing hypoxia. These findings could have implications for improving vascular-targeted therapy.

Objectives Lung cancer is the leading cause of cancer related deaths in worldwide. Despite recent advances in treatment options, patient survival has not improved substantially due to lack of commonly expressed molecular targets and effective targeted therapeutics. Thus, better understanding of the biology of lung cancer and identification of novel therapeutic targets are urgently needed for development of highly effective molecularly targeted therapies. Materials and methods Viability, proliferation and metastatic ability of lung cancer cells were evaluated using methylthiazoltetrazolium (MTT), colony formation and matrigel invasion assays, respectively. Western blotting, RT-PCR, and gene knockdown by siRNA transfections were carried out to investigate the effects of eEF-2K on lung cancer cells. Athymic Nu/Nu mice were treated with liposomal eEF-2KeEF-2K or control siRNA and tumor growth was evaluated in tumor xenograft models of lung cancer. Results and discussion Here, we report that Eukaryotic Elongation Factor-2 kinase (eEF-2K), a member of an atypical alpha kinases family, is significantly upregulated in lung cancer cell lines and its expression is associated with shorter overall patient survival in lung cancer. Inhibition eEF-2K expression by siRNA or a chemical inhibitorsignificantly suppressed lung cancer cell proliferation, colony formation, survival, migration/invasion and tumorigenesis by inhibiting cyclin D1, Src and Mitogen-Activated Protein Kinases/Extracellular Signal-Regulated Kinase (MAPK/ERK) signaling. In vivo targeting of eEF-2K by systemically injected nanoliposomal eEF-2K siRNA resulted in a significant inhibition of lung cancer tumor xenografts in nude mice. Our results suggest, for the first time, that expression of eEF-2K is associated with poor patient prognosis and involved in regulation of critical pathways, including Src and MAPK/ERK and cyclin D1, promoting tumor growth and progression, and thus may be a novel potential therapeutic target in lung cancer.