c-Myc plays a major role in the maintenance of glycolytic metabolism and hematopoietic stem cell (HSC) quiescence. Targeting modulators of HSC quiescence and metabolism could lead to HSC cell cycle entry with concomitant expansion. Here we show that c-Myc inhibitor 10074-G5 treatment leads to 2-fold increase in murine LSKCD34low HSC compartment post 7 days. In addition, c-Myc inhibition increases CD34+ and CD133+ human HSC number. c-Myc inhibition leads to downregulation of glycolytic and cyclin-dependent kinase inhibitor (CDKI) gene expression ex vivo and in vivo. In addition, c-Myc inhibition upregulates major HDR modulator Rad51 expression in hematopoietic cells. Besides, c-Myc inhibition does not alter proliferation kinetics of endothelial cells, fibroblasts or adipose derived mesenchymal stem cells, however; it limits bone marrow derived mesenchymal stem cell proliferation. We further demonstrate that a cocktail of c-Myc inhibitor 10074-G5 along with tauroursodeoxycholic acid (TUDCA) and i-NOS inhibitor L-NIL provides a robust HSC maintenance and expansion ex vivo as evident by induction of all stem cell antigens analyzed. Intriguingly, the cocktail of c-Myc inhibitor 10074-G5, TUDCA and L-NIL improves HDR related gene expression. These findings provide tools to improve ex vivo HSC maintenance and expansion, autologous HSC transplantation and gene editing through modulation of HSC glycolytic and HDR pathways.
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 β-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.
Meis1, which belongs to TALE-type class of homeobox gene family, appeared as one of the key regulators of hematopoietic stem cell (HSC) self-renewal and a potential therapeutical target. However, small molecule inhibitors of MEIS1 remained unknown. This led us to develop inhibitors of MEIS1 that could modulate HSC activity. To this end, we have established a library of relevant homeobox family inhibitors and developed a high-throughput in silico screening strategy against homeodomain of MEIS proteins using the AutoDock Vina and PaDEL-ADV platform. We have screened over a million druggable small molecules in silico and selected putative MEIS inhibitors (MEISi) with no predicted cytotoxicity or cardiotoxicity. This was followed by in vitro validation of putative MEIS inhibitors using MEIS dependent luciferase reporter assays and analysis in the ex vivo HSC assays. We have shown that small molecules named MEISi-1 and MEISi-2 significantly inhibit MEIS-luciferase reporters in vitro and induce murine (LSKCD34 l°w cells) and human (CD34 + , CD133 + , and ALDH hi cells) HSC self-renewal ex vivo. In addition, inhibition of MEIS proteins results in downregulation of Meis1 and MEIS1 target gene expression including Hif-1α, Hif-2α and HSC quiescence modulators. MEIS inhibitors are effective in vivo as evident by induced HSC content in the murine bone marrow and downregulation of expression of MEIS target genes. These studies warrant identification of first-in-class MEIS inhibitors as potential pharmaceuticals to be utilized in modulation of HSC activity and bone marrow transplantation studies. Meis1 is a member of TALE class of transcription factors 1. Through interaction domains in the N terminus, MEIS1 cooperates in transcription factors PBX1 and HOXA9 to transactivate target genes 2,3. MEIS2 and MEIS3 protein sequences demonstrate a high degree of similarity with MEIS1 4. Meis1 was first described in leukemia mouse model and identified as a viral integration site (reviewed in 5). MEIS proteins are characterized by PBX interaction domains and a highly conserved homeodomain (HD). MEIS1 HD shares identical MEIS2 HD amino acid sequence. Studies to understand how MEIS1 HD interacts with DNA led to crystallization of MEIS1 HD with target DNA and identification of DNA sequence preferentially bound by MEIS proteins as "TGACAG" 6-8. Meis1 is highly expressed in the bone marrow 2,9. Lethality occurs in Meis1 knockout mice at mid gestation (E14.5-15.5) with a number of hematopoietic, vascular and cardiac abnormalities 10-12. Conditional and tissue specific deletion of Meis1 in bone marrow led to loss of HSC quiescence associated with expansion of HSC pool in vivo 13. Meis1 has been shown to regulate HSC metabolism through transcriptional regulation of hypoxia factors including Hif1a and Hif2a 13-16. Deletion of Meis1 or Hif-1α in HSCs leads to reduction in the cytoplasmic glycolysis and induction of mitochondrial phosphorylation. Intriguingly, studies showed a fundamental role of Meis1 in neonatal cardiac regeneration. Increased Me...
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.
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.
BackgroundAlthough 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.MethodsFourteen 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.ResultsWith 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.ConclusionsOur 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3198-4) contains supplementary material, which is available to authorized users.
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