Novel analytic tools are needed to elucidate the molecular basis of leukemia-relevant gene mutations in the post-genome era. We generated isogenic leukemia cell clones in which the FLT3 gene was disrupted in a single allele using TALENs. Isogenic clones with mono-allelic disrupted FLT3 were compared to an isogenic wild-type control clone and parental leukemia cells for transcriptional expression, downstream FLT3 signaling and proliferation capacity. The global gene expression profiles of mutant K562 clones and corresponding wild-type controls were compared using RNA-seq. The transcriptional levels and the ligand-dependent autophosphorylation of FLT3 were decreased in the mutant clones. TALENs-mediated FLT3 haplo-insufficiency impaired cell proliferation and colony formation in vitro. These inhibitory effects were maintained in vivo, improving the survival of NOD/SCID mice transplanted with mutant K562 clones. Cluster analysis revealed that the gene expression pattern of isogenic clones was determined by the FLT3 mutant status rather than the deviation among individual isogenic clones. Differentially expressed genes between the mutant and wild-type clones revealed an activation of nonsense-mediated decay pathway in mutant K562 clones as well as an inhibited FLT3 signaling. Our data support that this genome-editing approach is a robust and generally applicable platform to explore the molecular bases of gene mutations. Overall design: Global gene expression profiles of three isogenic K562 mutant clones (clones k20, k112, k324) and three randomly selected wild-type clones (clones kw1, kw2, kw3) were generated by RNA-seq, using Illumina Hiseq 2000.
TALENs-mediated gene disruption of FLT3 in leukemia cells: Using genome-editing approach for exploring the molecular basis of gene abnormality.
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To design an effective antibody therapy to improve clinical outcomes in leukemia, the identification of novel cell surface antigens is needed. Herein, we demonstrate a role for transmembrane tumor necrosis factor-in leukemia. To characterize tmTNF- expression in acute leukemia, normal hematopoietic cells, and non-hematopoietic tissues, we used a monoclonal antibody, termed C1, which specifically recognizes the tmTNF- domain. We found that tmTNF- was preferentially expressed by acute leukemia and leukemia stem cells. More abundant expression correlated with poor risk stratification, extramedullary infiltration, and adverse clinical parameters. Moreover, knockdown of tmTNF-+ expression rendered leukemia cells more sensitive to chemotherapy in vitro and delay regeneration of leukemia in NODSCID mice. Targeting tmTNF- by C1 resulted in leukemia cell killing via antibody-dependent cell-mediated and complement -dependent cytotoxicity in vitro, and inhibited leukemia cell growth in vivo while simultaneously sparing normal hematopoietic cells. Notably, C1 administration impaired the regeneration of leukemia in secondary serial transplantationin to NOD-SCID mice.
Transmembrane TNF-α preferentially expressed by leukemia stem cells and blasts is a potent target for antibody therapy.
Specimen part, Cell lineView Samples
The pancreatic beta cell function failure is the core event of type 2 diabetes mellitus. High levels of free fatty acid and glucose are two main factor that induced pancreatic beta cell function failure. Long term exposure to palmitate can induced pancreatic beta cell apoptoss and impaired insulin secretion in vivo and in vitro, called lipotoxicity. The lipotoxicity often coupled with high glucose, their combination form called glucolipotoxcity. We carried out temporal transcriptome and proteome studies investigating the evolution of molecular events in Ins1 cells stimulated by palmitate for different times. And through compared the transcriptome and proteome between lipotoxicity and glucolipotoxicity explain the mechanism of glucolipotoxicity more harmfull to beta cell. Overall design: we performed a time-course large-scale transcriptome of INS1 cells induced by 0.5 mM palmitate and 0.5 mM palmitate with 27.8 mM glucose at four time points (8, 16, 24 and 48 h) using MAPS method
Temporal Proteomic Analysis of Pancreatic β-Cells in Response to Lipotoxicity and Glucolipotoxicity.
Specimen part, Cell line, Subject, TimeView Samples
Little is known about the roles of methyl-CpG-binding domain protein 2 (MBD2), a reader of DNA methylation, in T-cell acute lymphoblastic leukemia (T-ALL). Here, we investigated the role of MBD2 in T-ALL by using an Mbd2 knockout mouse model. We found that MBD2 ablation impeded the progression and maintenance of Notch1-driven T-ALL.Our data reveals essential roles for MBD2 in lymphopoiesis and T-ALL and support an intriguing potential of MBD2 as a therapeutic target for T-ALL.
MBD2 Ablation Impairs Lymphopoiesis and Impedes Progression and Maintenance of T-ALL.
Specimen partView Samples
Compare with normal nasopharyngeal epithelial cells, we found ACAT1 was decreased in NPC cells, we found that ACAT1 inhibited proliferation, colony formation, migration and invasion in NPC cells. We used microarrays to identify differential genes regulated by ACAT1 in NPC cell lines.
Epigenetic Inactivation of Acetyl-CoA Acetyltransferase 1 Promotes the Proliferation and Metastasis in Nasopharyngeal Carcinoma by Blocking Ketogenesis.
Cell line, TreatmentView Samples
Prostate cancer is the most common cancer in men and cardiac glycosides inhibit prostate cancer cell proliferation. In order to investigate the mechanism by which cardiac glycosides inhibit prostate cancer cells, we observed genome-wide RNA expression in prostate cancer LNCaP-abl cells, hormone resistant cells, after the cardiac glycoside treatment using RNA-Seq. In addition, we profiled LNCaP-abl cells after androgen receptor (AR) knockdown to observe whether cardiac glycoside effect on RNA expression is similar to that of AR knockdown. Overall design: Observation of three cardioglycosides, Digoxin, Peruvoside and Strophanthidin, and AR knockdown regulated RNA expression in LNCaP-abl with RNA-Seq (each triplicates)
Versatile pathway-centric approach based on high-throughput sequencing to anticancer drug discovery.
Cell line, SubjectView Samples
Direct conversion of somatic cells into neurons holds great promise for regenerative medicine. However, neuronal conversion is relatively inefficient in human cells compared to mouse cells. It has been unclear what might be the key barriers to reprogramming in human cells. We recently elucidated an RNA program mediated by the polypyrimidine tract binding protein PTB to convert mouse embryonic fibroblasts (MEFs) into functional neurons. In human adult fibroblasts (HAFs), however, we unexpectedly found that invoking the documented PTB–REST–miR-124 loop generates only immature neurons. We now report that the functionality requires sequential inactivation of PTB and the PTB paralog nPTB in HAFs. Inactivation of nPTB triggers another self-enforcing loop essential for neuronal maturation, which comprises nPTB, the transcription factor BRN2, and miR-9. These findings suggest that two separate gatekeepers control neuronal conversion and maturation and consecutively overcoming these gatekeepers enables deterministic reprogramming of HAFs into functional neurons. Overall design: Six RNA-seq libraries are generated by MAPS approach. Total RNA is extracted from induced neuronal cells derived from control shRNA or PTB shRNA treated human adult fibroblasts. Please note that the ''RNA-seq HAF hygro'' sample was on 6 days after switching to N3 media *without* nPTB depletion. The other two ''shPTB 6d'' and ''shPTB 3w'' were on 6 days and 3 weeks respectively after switching to N3 media *with* nPTB depletion.
Sequential regulatory loops as key gatekeepers for neuronal reprogramming in human cells.
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AHI-1 is an oncogene often targeted by provirus insertional mutagenesis in murine leukemias and lymphomas. Aberrant expression of human AHI-1 occurs in cutaneous T-cell lymphoma (CTCL) cells and in CD4+CD7- Sezary cells from patients with Sezary syndrome (SS). Stable knockdown of AHI-1 using retroviral-mediated RNA interference in CTCL cells inhibits their transforming activity in vitro and in vivo. To identify genes involved in AHI-1-mediated transformation, microarray analysis was performed to identify differentially expressed genes in AHI-1 suppressed CTCL cells. Fifteen up-regulated and six down-regulated genes were identified and confirmed by Q-RT-PCR. Seven were further confirmed in a microarray analysis of CD4+CD7- Sezary cells from SS patients. HCK and BIN1 emerged as new candidate cooperative genes, with differential protein expression which correlates with observed transcript changes. Interestingly, changes in HCK phosphorylation and biological response to its inhibitor, dasatinib, were observed in AHI-1 suppressed or overexpressed cells. The tumor suppressor BIN1 physically interacts with MYC in CTCL cells, which also exhibit differential MYC protein expression. In addition, aberrant expression of alternative splicing forms of BIN1 was observed in primary and transformed CTCL cells. These findings indicate that HCK and BIN1 may play critical roles in AHI-1-mediated leukemic transformation of human CTCL cells.
Identification of tyrosine kinase, HCK, and tumor suppressor, BIN1, as potential mediators of AHI-1 oncogene in primary and transformed CTCL cells.
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