Project description:Cell-specific gene expression is achieved by a combination of mechanisms including transcriptional and post-transcriptional regulation. The transcription factor Nkx2-1, essential for lung cell differentiation, mainly acts in transcriptional activation but can directly or indirectly repress gene expression. microRNAs are a class of small non-coding RNA that control one of the major mechanisms of gene repression. To identify miRNAs regulated by Nkx2-1 that may mediate its repressing effects, we knocked-down Nkx2-1 in mouse lung epithelial cell lines and systematically identified targets by genome-wide miR and mRNA expression analyses. Nkx2-1 controls expression of miRs known to contribute to lung cell differentiation in development and disease and others not previously described. Amongst the significantly altered miRs, the mir-106a-363 cluster, miR-1195, miR-378, and miR-346 are directly correlated with the levels of Nkx2-1, whereas miR-200c/b, miR-221, and miR- 222 are inversely correlated. These miRNAs are expressed in embryonic lung at day E11.5, and/or E19.5 determined by in-situ hybridization. Expression of predicted targets of mir-1195, mir-346 and miR-200c and mir-221/222 were evaluated by mRNA expression microarrays in Nkx2-1 knockdown cells identifying those anti-correlated to the corresponding miRNA expression. Genes regulated by mir-1195, Cyp2s1 and Map3k2, by mir-346, Klf6, and miR-200c, Myb, Nfib, and Six1, were validated by qRT-PCR. Inhibition of mir-1195 confirms the inverse correlation of this miRNA with its putative targets Cyp2s1 and Map3k2. This miRNA-mRNA expression analysis identifies potential paths of Nkx2-1 mediated gene repression, and contributes to the understanding of gene regulation in lung epithelial differentiation and development. Nkx2-1 mRNA was knocked down in lung epithelial cells using a lentivirus expressing a shRNA targeting Nkx2-1 (n=3) and compared to empty vector controls (n=3).
Project description:To gain insights whether and if activation of the nuclear receptor FXR is involved in the disease ethiology of ATP8B1 deficiency, we contstructed a Caco-2 ATP8B1 knock down cell line and a Caco-2 empty vector control cell line. Furthermore we expect to gain new insights from transcriptional profiling of ATP8B1 deficient cells.
Project description:CASPASE-3 (CASP3) is well known for its proteolytic function that mediates multiple key cell death-initiated processes and other related cellular processes. However, the possibility that CASP3 may also possess important additional non-catalytic functions in mammalian cells has remained largely unexplored. We now report the results of CASP3 knockdown, rescue, proteomic analysis and flow cytometry experiments initially in normal and malignant human mammary cells and later shown to extent to all other human cell types tested. The results reveal a new role of the CASP3 prodomain in regulating the cell cycle progression, survival, proliferation, and protein aggregate accumulation in all cells tested. The generality of these findings suggests that the ancestral pro-survival role of CASP3 in yeast persisted throughout evolution via a conservation of its prodomain, predating its later acquisition of an inherent catalytic property and subsequently preserved cell death control functions.
Project description:To test whether or not the stable restoration of PMEL and FHL2 knockdown affects the oncogenic ability of into amelanotic melanoma cells. Global gene expression analysis performed to identify what different mechanisms and/or factors might be involved in cell motility and cell aggressiveness. A375M2 were stable transduced with lentivirus containing an empty vector expressing eGFP, full length PMEL or shRNA specific sequence against FHL2. Cells were FACS selected and grown in selective medium for 15 days before testing. Total RNA was extracted from cells after being seeded at equal numbers and reached 80% confluence. Samples were obtained from three separate experiments and send together for processing.
Project description:Herpesviruses are known to encode micro (mi)RNAs and to use them to regulate the expression of both viral and cellular genes. The genome of Kaposi’s sarcoma herpesvirus (KSHV) encodes a cluster of twelve miRNAs, which are abundantly expressed during both latency and lytic infection. Relatively few cellular targets of KSHV miRNAs are known. Here, we used a microarray expression profiling approach to analyze the transcriptome of both B lymphocytes and endothelial cells stably expressing KSHV miRNAs and monitor the changes induced by the presence of these miRNAs. We generated a list of potential cellular targets by looking for miRNA seed-match-containing transcripts that were significantly down regulated upon KSHV miRNAs expression. Interestingly, the overlap of putative targets identified in B lymphocytes and endothelial cells was minimal, suggesting a tissue-specific target-regulation by viral miRNAs. Among the putative targets, we identified caspase 3, a critical factor for the control of apoptosis, which we validated using luciferase reporter assays and western blotting. In functional assays we obtained further evidence that KSHV miRNAs indeed protect cells from apoptosis. Single-channel hybridization to Affymetrix oligonucleotides microarrays. DG75 and EA.hy926 cells were transducted in triplicates with the K10/12 and K12/12 lentiviral constructs expressing the KSHV miRNAs, or with a GFP-expressing construct as control. The K10/12 construct expressed only the ten intronic miRNAs miR-K12-1 to 9 and miR-K12-11 while the K12/12 construct also expressed miR-K12-10 and miR-K12-12 in addition.
Project description:MLK3 gene mutations were described to occur in about 20% of microsatellite unstable gastrointestinal cancers and to harbor oncogenic activity. In particular, mutation P252H, located in the kinase domain, was found to have a strong transforming potential, and to promote the growth of highly invasive tumors when subcutaneously injected in nude mice. Nevertheless, the molecular mechanism underlying the oncogenic activity of P252H mutant remained elusive.In this work, we performed Illumina Whole Genome arrays on three biological replicas of human HEK293 cells stably transfected with the wild-type MLK3, the P252H mutation and with the empty vector (Mock) in order to identify the putative signaling pathways associated with P252H Our results showed that mutant MLK3 exerts its oncogenic effects by deregulating several important colorectal cancer- associated signaling pathways such as WNT, MAPK, NOTCH, TGF-M-NM-2 and P53. A more detailed analysis of the alterations affecting the WNT signaling pathway revealed a down-regulation of molecules involved in the canonical pathway, such as DVL2, LEF1, CCND1 and c-Myc, and an up- regulation of DKK, a well-known negative regulator of canonical WNT signaling, in MLK3 mutant cells. Additionally, FZD6 and FZD10 genes, known to act as negative regulators of the canonical WNT signaling cascade and as positive regulators of the planar cell polarity (PCP) pathway, a non-canonic WNT pathway, were found to be up-regulated in P252H cells. 9 samples, 2 conditions with 3 biological replicates per condition
Project description:Translocation renal cell carcinoma (tRCC) is a rare, aggressive kidney cancer primarily occurring in children. They are genetically defined by translocations involving MiT/TFE gene family members, TFE3. We utilized human kidney organoids, or tubuloids, to engineer a tRCC model by expressing of one of the most common MiT/TFE fusions, SFPQ-TFE3. Lentiviral transductions were performed as previously described with lentiviruses encoding either pLKO.1-UbC-luciferase-blast (TubCtrl), pLKO.1-UbC-TFE3-blast (TubTFE3), or pLKO.1-UbC-SFPQ-TFE3-blast (TubFus). Two days post transduction, 5 µg/ml blasticidin was added to the culture medium to select for successfully transduced cells. To study the genome-wide binding sites of the fusion, we conducted CUT&RUN sequencing. CUT&RUN experiments were performed using a modified protocol for low cell numbers using the following antibodies: anti-TFE3 (ab93808, Abcam, 1:2000). Libraries were sequenced using an Illumina NextSeq2000 (2x100bp).
Project description:In this study, we discover an essential role for the mitochondrial glutamate-aspartate antiporter SLC25A13 in biomass synthesis in endothelial cells (ECs). To delineate the global changes of SLC25A13 silencing at a molecular level, we performed high-throughput bulk RNA-sequencing and analyzed the differences between the gene expression profiles of CTRL and SLC25A13-KD ECs. HUVECs were transduced at a multiplicity of infection (MOI) of 30. Transductions were performed on day 0 and after 24 hours the cells were refed with fresh medium. RNA of scrambled control and SLC25A13-KD ECs were extracted 6 days after seeding. Samples were indexed to allow for multiplexing. Libraries were sequenced on an Illumina NovaSeq6000 platform. Single-end reads of 100 bp length were produced with a minimum of 20 M reads per sample. The reads were aligned using a standard pipeline for splice-aware alignment. The reads per gene were then quantified. The study accounted for 12 samples (6 for CTRL and 6 for SLC25A13-KD ECs).
Project description:Developmental checkpoints in stem/progenitor cells are critical to the determination, commitment and differentiation into distinct lineages. Cancer cells often retain expression of lineage-specific checkpoint proteins, but their potential impact in cancer remains elusive. T lymphocytes mature in the thymus following a highly orchestrated developmental process that entails the successive rearrangements and expression of T-cell receptor (TCR) genes. Low affinity recognition of self-peptide/MHC complexes (self-pMHC) presented by thymic epithelial cells by the TCR of CD4+CD8+ (DP) cortical thymocytes transduces positive selection signals that ultimately shape the developing T cell repertoire. DP thymocytes not receiving these signals die by lack of stimulation whereas those that recognize self-pMHC with high affinity undergo TCR-mediated apoptosis and negative selection. In T-cell acute lymphoblastic leukaemia (T-ALL), leukaemic transformation of maturating thymocytes results from the acquisition of multiple genetic and epigenetic alterations in oncogenes and tumour suppressor genes, that disrupt the normal regulatory circuits and drive clonal expansion of differentiation-arrested lymphoblasts. We show here that TCR triggering by negatively-selecting self-pMHC prevented T-ALL development and leukaemia maintenance in mice. Induction of TCR signalling by high affinity self-pMHC or treatment with monoclonal antibodies to the CD3 signalling chain (anti-CD3) caused massive leukaemic cell death and a gene expression program resembling that of thymocyte negative selection. Importantly, anti-CD3 treatment hampered leukaemogenesis in mice transplanted with either mouse or patient-derived T-ALLs. These data provide a rationale for targeted therapy based on anti-CD3 treatment of T-ALL patients and demonstrate that endogenous developmental checkpoint proteins are amenable to therapeutic intervention in cancer cells. Gene expression data from four culture conditions was performed for the following cells: ALL-SIL-TCRα/β-GFP co-cultured on OP9-DL1 for 48 h, ALL-SIL-TCRα/β-GFP without co-culture, ALL-SIL cells transduced with TLX shRNA (sh-TLX), and ALL-SIL transduced with sh-control vectors. All conditions are performed in two replicates.