miRNAs as cell fate determinants of lateral and paraxial mesoderm differentiation from embryonic stem cells.

To date, the role of miRNAs on pluripotency and differentiation of ESCs into specific lineages has been studied extensively. However, the specific role of miRNAs during lateral and paraxial mesoderm cell fate decision is still unclear. To address this, we firstly determined miRNA profile of mouse ESCs differentiating towards lateral and paraxial lineages which were detected using Flk1 and PDGFαR antibodies, and of myogenic and hematopoietic differentiation potential of purified paraxial and lateral mesodermal cells within these populations. miRNAs associated with lateral and paraxial mesoderm, and their targets were identified using bioinformatics tools. The targets of the corresponding miRNAs were validated after transfection into mouse ESCs. The roles of the selected miRNAs in lateral, and paraxial mesoderm formation were assessed along with hematopoietic and myogenic differentiation capacity. Among the miRNAs, mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p, upregulated in lateral mesoderm cells, and mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p, upregulated in paraxial mesoderm cells. While transient co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p increased the number of lateral mesodermal cells, co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p increased the number of paraxial mesodermal cells. Moreover, differentiation potential of the lateral mesodermal cells into hematopoietic cell lineage increased upon co-transfection of mmu-miR-126a-3p, mmu-miR-335-5p and mmu-miR-672-5p and differentiation potential of the paraxial mesodermal cells into skeletal muscle lineage were increased upon co-transfection of mmu-miR-10b-5p, mmu-miR-196a-5p and mmu-miR-615-3p. In conclusion, we determined the miRNA profile of lateral and paraxial mesodermal cells and co-transfection of miRNAs increased differentiation potential of both lateral and paraxial mesodermal cells transiently.

The role of TNF-α in chordoma progression and inflammatory pathways.

PURPOSE: Chordomas are highly therapy-resistant primary bone tumors that exhibit high relapse rates and may induce local destruction. Here, we evaluated the effects of tumor necrosis factor-alpha (TNF-α) on chordoma progression and clinical outcome. METHODS: Chordoma cells were treated with TNF-α after which its short- and long-term effects were evaluated. Functional assays, qRT-PCR and microarray-based expression analyses were carried out to assess the effect of TNF-α on chemo-resistance, epithelial to mesenchymal transition (EMT), migration, invasion and cancer stem cell-like properties. Finally, relationships between TNF-α expression and clinicopathological features were assessed in a chordoma patient cohort. RESULTS: We found that TNF-α treatment increased the migration and invasion of chordoma cells. Also, NF-κB activation was observed along with increased EMT marker expression. In addition, enhanced tumor sphere formation and soft agar colony formation were observed, concomitantly with increased chemo-resistance and CD338 marker expression. The TNF-α and TNFR1 expression levels were found to be significantly correlated with LIF, PD-L1 and Ki67 expression levels, tumor volume and a short survival time in patients. In addition, a high neutrophil to lymphocyte ratio was found to be associated with recurrence and a decreased overall survival. CONCLUSIONS: From our data we conclude that TNF-α may serve as a prognostic marker for chordoma progression and that tumor-promoting inflammation may be a major factor in chordoma tumor progression.

Distinctive role of dysregulated miRNAs in chordoma cancer stem-like cell maintenance.

Chordoma is a rare, slow-growing tumor thought to arise from remnants of embryonic notochord associated with an aggressive outcome. Cancer stem-like cells (CSCs) are related to tumorigenesis, recurrence, and resistance in cancers. Therefore, chordoma CSCs are possible targets for chordoma treatment. In this study, dysregulated miRNAs were determined in chordoma CSCs and identified their role in chordoma. Dysregulated miRNAs were determined via miRNA microarray and validated through qPCR. miRNAs were transiently transfected to the chordoma cell lines and their roles in proliferation, apoptosis, migration and invasion capacities and stem-like properties were identified. Finally, a relationship between clinicopathological features and dysregulated miRNAs has been evaluated among 21 chordoma patients. CD133+CD15+ cells exhibited CSC phenotype with increased CSC- and Epithelial-Mesenchymal Transition (EMT)-related gene expression, invasion, migration, tumorosphere- and colony-forming abilities. In addition, WNT5A, TGF-α, BTG2 and MYCBP genes involved in CSC-related pathways, were targets of miR-140-3p, miR-148a-3p, miR-210-5p and miR-574-5p, respectively. Transfection of CSC-related miRNAs also increased migration and invasion along with stem cell phenotype. Finally, we determined that miR-140-3p and miR-148a-3p expressions correlated with Ki67 while miR-140-3p and TGF-α expressions were correlated with p53. Moreover, MYCBP expression was positively correlated with tumor volume, and metastasis was associated with the expression of miR-210-5p and TGF-α in our patient cohort. Through these findings, we conclude that chordoma CSCs have distinctive miRNA profile, which can regulate stem-like properties of chordoma CSCs.

Simultaneous miRNA and mRNA transcriptome profiling of glioblastoma samples reveals a novel set of OncomiR candidates and their target genes.

Although glioblastomas are common, there remains a need to elucidate the underlying mechanisms behind their initiation and progression and identify molecular pathways for improving treatment. In this study, sixteen fresh-frozen glioblastoma samples and seven samples of healthy brain tissues were analyzed with miRNA and whole transcriptome microarray chips. Candidate miRNAs and mRNAs were selected to validate expression in fifty patient samples in total with the criteria of abundance, relevance and prediction scores. miRNA and target mRNA relationships were assessed by inhibiting selected miRNAs in glioblastoma cells. Functional tests have been conducted in order to see the effects of miRNAs on invasion, migration and apoptosis of GBM cells. Analyses were carried out to determine correlations between selected molecules and clinicopathological features. 1332 genes and 319 miRNAs were found to be dysregulated by the microarrays. The results were combined and analyzed with Transcriptome Analysis Console 3 software and the DAVID online database. Primary differential pathways included Ras, HIF-1, MAPK signaling and cell adhesion. OncomiR candidates 21-5p, 92b-3p, 182-5p and 339-5p for glioblastoma negatively correlated with notable mRNA targets both in tissues and in in vitro experiments. miR-21-5p and miR-339-5p significantly affected migration, invasion and apoptosis of GBM cells in vitro. Significant correlations with overall survival, tumor volume, recurrence and age at diagnosis were discovered. In this article we present valuable integrated microarray analysis of glioblastoma samples regarding miRNA and gene-expression levels. Notable biomarkers and miRNA-mRNA interactions have been identified, some of which correlated with clinicopathological features in our cohort.

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fibroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Leukemia Inhibitory Factor Promotes Aggressiveness of Chordoma.

Chordomas are rare tumors of the spine and skull base that are locally destructive and resistant to chemotherapy and radiation therapy, with a poor prognosis and limited therapeutic options. Chordoma patients have a long life expectancy with high mortality from the disease. Cancer stem cells, which are known to exist in chordomas, have extensive proliferative and self-renewal potential and are responsible for maintaining tumor heterogeneity along with chemotherapy and radiotherapy resistance. Leukemia inhibitory factor (LIF) has multiple functions in stem cell biology, the immune response, and cancer, and is potentially a key molecule that allows cancer stem cells to self-renew. The purpose of this study was to determine whether LIF increases the aggressive traits of chordoma cells and leads to a poor prognosis in patients. Chordoma cell lines were treated with LIF, and functional tests were done. Twenty skull base chordoma samples were checked for levels of LIF and a correlation with clinicopathological features. The whole transcriptome microarray was used to observe changes in gene expression. We observed increased migration, invasion, tumorosphere formation, colony formation, epithelial-mesenchymal transition, and chemoresistance accompanied by a dramatic elevation in inflammatory gene networks and pathways in chordomas. The expression of LIF was associated with tumor size and a poorer overall survival. Microarray and quantitative real-time polymerase chain reaction assessments suggest that LIF can facilitate tumor-promoting inflammation. Results indicate that LIF plays a role in maintaining cancer stem cells in chordomas.

A new hope for obesity management: Boron inhibits adipogenesis in progenitor cells through the Wnt/ß-catenin pathway.

Obesity is a worldwide medical problem resulting in serious morbidity and mortality involving differentiation of pre-adipocytes into mature adipocytes (adipogenesis). Boron treatment has been reported to be associated with weight reduction in experimental animals; however, its effects on pre-adipocyte differentiation and anti-adipogenic molecular mechanisms are unknown. In this study, we demonstrate the inhibitory activities of boric acid (BA) and sodium pentaborate pentahydrate (NaB) on adipogenesis using common cellular models. Boron treatment repressed the expression of adipogenesis-related genes and proteins, including CCAAT-enhancer-binding protein α and peroxisome proliferator-activated receptor γ, by regulating critical growth factors and the ß-catenin, AKT, and extracellular signal-regulated kinase signaling pathways. In addition, although boron treatment did not induce apoptosis in pre-adipocytes, it depressed mitotic clonal expansion by regulation of cell cycle genes. Overall, these data offer promising insights into the prevention/treatment of obesity and associated diseases.

Simultaneous analysis of miRNA-mRNA in human meningiomas by integrating transcriptome: A relationship between PTX3 and miR-29c.

BACKGROUND: Although meningioma is a common disease, there is a lack of understanding of the underlying molecular mechanisms behind its initiation and progression. We used combined miRNA-mRNA transcriptome analysis to discover dysregulated genes and networks in meningiomas. METHODS: Fourteen fresh-frozen meningioma samples and one human meningeal cell line were analyzed by using miRNA and whole transcriptome microarray chips. Data was filtered and analyzed. Candidate miRNAs and mRNAs were selected for validation in fifty-eight patient samples. miRNA and target mRNA relationships were assessed by inhibiting miRNA in meningioma cells. Apoptosis and viability assays were also used as functional tests. RESULTS: With the whole transcriptome microarray, 3753 genes were found to be dysregulated, and 891 miRNAs were found to be dysregulated as a result of miRNA microarray. Results were combined and analyzed with bioinformatics tools. Top differential pathways included those of inflammation, cancer, and cellular growth and survival. The oncosupressor PTX3 was constitutively low in meningioma samples. Moreover, PTX3 negatively correlated with miR-29c in our samples. Inhibiting miR-29c upregulated the PTX3 level, induced apoptosis of meningioma cells, and decreased cell viability. CABIN1, miR-29c, TMOD1, PTX3, RPL22, SPARCL1 and RELA were correlated with clinicopathological features in patient samples. CONCLUSIONS: Our results present the first integrated mRNA-miRNA analysis in meningiomas. miR-29c-3p and PTX3 are inversely correlated in tissues and meningioma cells, hinting that PTX3 can be regulated by miR-29c-3p. Furthermore, we determined potential clinicopathological markers.

The potential function of microRNA in chordomas.

Little is known about the molecular biology of chordomas, which are rare, chemoresistant tumors with no well-established treatment. miRNAs regulate gene networks and pathways. We aimed to evaluate the effects of dysregulated miRNA in chordomas would help reveal the underlying mechanisms of chordoma initiation and progression. In this study, miR-31, anti-miR-140-3p, anti-miR148a, and miR-222 were transiently transfected to chordoma cell lines and an MTS assay, apoptosis assay, and cell-cycle analysis were conducted to evaluate the effects. The mRNA level of predicted and confirmed targets of each miRNA, as well as the EMT and MET markers of U-CH1 and MUG-Chor1, were assessed with real-time polymerase chain reaction. Transient transfection of miRNA mimics was achieved, as each mimic increased or decreased the level of its corresponding miRNA. miR-31 decreased cell viability in MUG-Chor1 and U-CH2 after 72h, which is consistent with previous findings for U-CH1. Both miR-31 and anti-miR-148a induced apoptosis in all three cell lines. Although each miRNA had a similar pattern, miR-31 had the most effective S-phase arrest in all three cell lines. RDX, MET, DNMT1, DNMT3B, TRPS1, BIRC5, and KIT were found to be targeted by the selected miRNAs. The level of miR-222 in chordoma cell lines U-CH1 and MUG-Chor1 correlated positively with EMT markers and negatively with MET markers. This study uncovered the potential of miR-31, miR-140-3p, miR-148a, and miR-222-3p to be key molecules in the cell viability, cell cycle, and apoptosis in chordomas, as well as initiation, differentiation, and progression.

The molecular aspects of chordoma.

Chordomas are one of the rarest bone tumors, and they originate from remnants of embryonic notochord along the spine, more frequently at the skull base and sacrum. Although they are relatively slow growing and low grade, chordomas are highly recurrent, aggressive, locally invasive, and prone to metastasize to the lungs, bone, and the liver. Chordomas highly and generally show a dual epithelial-mesenchymal differentiation. These tumors resist chemotherapy and radiotherapy; therefore, radical surgery and high-dose radiation are the most used treatments, although there is no standard way to treat the disease. The molecular biology process behind the initiation and progression of a chordoma needs to be revealed for a better understanding of the disease and to develop more effective therapies. Efforts to discover the mysteries of these molecular aspects have delineated several molecular and genetic alterations in this tumor. Here, we review and describe the emerging insights into the molecular landscape of chordomas.

Familial Mediterranean fever gene mutations in north-eastern part of Anatolia with special respect to rare mutations.

We aimed to determine the frequency of mutations, carrier rates and the association of rare mutations with Familial Mediterranean Fever (FMF) symptoms. There is a need to evaluate as many different populations as possible in order to determine either specific rare mutations or a range of disease-associated mutations. The demographic data and FMF symptoms related to MEFV gene mutations were collected from 731 participants. Exon 2 and exon 10 of the MEFV gene were tested by DNA sequencing. The rare mutations were identified as: M694I (1.1%, n=12), E148V (0.6%, n=6), T267I (0.5%, n=5), L110P (0.2%, n=2), E167D (0.2%, n=2), K695R (0.1%, n=1) and an insertion G (Guanine) mutation (0.4%, n=4) at the 777th codon of exon 10. We used routine comprehensive detection systems such as Sanger sequence that can catch rare mutations, for definite diagnosis and treatment of FMF disease.

Overexpression of miR-145-5p inhibits proliferation of prostate cancer cells and reduces SOX2 expression.

We aimed to perform functional analysis of miR-145-5p in prostate cancer (PCa) cells and to identify targets of miR-145-5p for understanding its role in PCa pathogenesis. PC3, DU145, LNCaP PCa, and PNT1a nontumorigenic prostate cell lines were utilized for functional analysis of miR-145-5p. Its overexpression caused inhibition of proliferation through apoptosis and reduced migration in PCa cells. SOX2 expression was significantly decreased in both mRNA and protein level in miR-145-5p-overexpressed PCa cells. We proposed that miR-145-5p, being an important regulator of SOX2, carries a crucial role in PCa tumorigenesis.

MicroRNA expression profiling reveals the potential function of microRNA-31 in chordomas.

Chordomas are rare bone tumors arising from remnants of the notochord. Molecular studies to determine the pathways involved in their pathogenesis and develop better treatments are limited. Alterations in microRNAs (miRNAs) play important roles in cancer. miRNAs are small RNA sequences that affect transcriptional and post-transcriptional regulation of gene expression in most eukaryotic organisms. Studies show that miRNA dysregulation is important for tumor initiation and progression. We compared the expression profile of miRNAs in chordomas to that of healthy nucleus pulposus samples to gain insight into the molecular pathogenesis of chordomas. Results of functional studies on one of the altered miRNAs, miR-31, are presented. The comparison between the miRNA profile of chordoma samples and the profile of normal nucleus pulposus samples suggests dysregulation of 53 miRNAs. Thirty miRNAs were upregulated in our tumor samples, while 23 were downregulated. Notably, hsa-miR-140-3p and hsa-miR-148a were upregulated in most chordomas relative to levels in nucleus pulposus cells. Two other miRNAs, hsa-miR-31 and hsa-miR-222, were downregulated in chordomas compared with the control group. Quantification with real-time polymerase chain reaction confirmed up or downregulation of these miRNAs among all samples. Functional analyses showed that hsa-miR-31 has an apoptotic effect on chordoma cells and downregulates the expression of c-MET and radixin. miRNA profiling showed that hsa-miR-31, hsa-miR-222, hsa-miR-140-3p and hsa-miR-148a are differentially expressed in chordomas compared with healthy nucleus pulposus. Our profiling may be the first step toward delineating the differential regulation of cancer-related genes in chordomas, helping to reveal the mechanisms of initiation and progression.

The effects of chemotherapeutic agents on differentiated chordoma cells.

OBJECT: Chordoma is a rare type of malignant bone tumor and is known to arise from the remnants of the notochord. Resistance to chemotherapy makes the treatment of chordoma difficult; therefore, new approaches need to be developed to cure this disease. Differentiation therapy, using various differentiating agents, is attracting oncologists as a common therapeutic method to treat other tumors. Based on forcing cells to mature into other lineages, differentiation therapy might be an available method to treat chordomas in addition to conventional therapies. METHODS: In this study a chordoma cell line, U-CH1, was exposed to several chemotherapeutic agents including vincristine, doxorubicin, cisplatin, etoposide, fludarabine, methotrexate, nilotinib, and imatinib mesylate under appropriate conditions. The first group of U-CH1 cells was exposed to drugs only and the second group of cells was exposed to the simultaneous treatment of 1 µM all-trans retinoic acid (ATRA) and chemotherapeutic agents in differentiation therapy. The efficacy of the differentiation method was assessed by measuring the viability of U-CH1 cells. RESULTS: Vincristine, doxorubicin, etoposide, cisplatin, and fludarabine, each at a concentration of 10 µM, decreased the number of chordoma cells when given alone down to 11%, 0%, 30%, 67%, and 3%, respectively. Etoposide and cisplatin, each at a concentration of 10 µM, reduced the percentage of viable chordoma cells in a more effective way when given with 1 µM ATRA simultaneously, reducing the number of viable cells to 14% and 9%, respectively. On the other hand, imatinib and nilotinib, each at a concentration of 3 µM, as well as 10 µM methotrexate, showed no decrease in the number of cancer cells. CONCLUSIONS: The results suggest that chordoma cells may be treated using the differentiation method in a more effective way than when they are treated with chemotherapeutic agents alone. This new approach may be an alternative method to conventional therapies in the treatment of chordoma.

RNA interference and amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a debilitating neuro-degenerative disorder characterized by progressive loss of motor neurons. The etiology and molecular pathogenesis of cell death in most sub-types of the disease are largely unknown. The best documented cause of moto-neuron degeneration is the mutation in the superoxide dismutase-1 (SOD1) gene, which occurs in 10% of the familial forms of ALS. Discovery of RNA interference (RNAi), which plays an important role in the regulation of gene expression, has proven to be a powerful tool to study the pathogenesis and to develop innovative treatment options for hereditary diseases, including familial variants of ALS. This review summarizes current research advances in RNAi in relation to ALS.