Transcriptional profiling of TSC2-null cell lines UMB1949 and 621-102 (TRI102)
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ABSTRACT: RNA-sequencing was performed to establish the transcriptional profiles of two cell lines used in our study. 621-102 (TRI102) were originally isolated from a lymphangioleiomyomatosis-associated renal angiomyolipoma. UMB1949 were originally isolated from a male tuberous sclerosis complex (TSC) patient with renal angiomyolipoma and immortalized via SV40 large T antigen and hTERT introduction. These samples were seuqenced as part of a larger study (Martin et al. 2017, Nat. Comm. https://doi.org/10.1038/ncomms15816).
Project description:RNA-seq profiling of Tsc2-deficient mouse embryonic fibroblasts (MEFs) and TSC2-deficient 621-101 human angiomyolipoma cells exposed to oleic acid or basal condition.
Project description:Transcriptome analysis of total RNA samples from human cell line (LAM 621-101, female) Global gene and splice isoform expression profiling was performed to get a comprehensive view of transcriptome changes in human Lymphangioleiomyomatosis (LAM 621-101, female) cell line after inhibition of mTORC1 and SRPK2 proteins.
Project description:Patients with Lymphangioleiomyomatosis (LAM) develop nodules of cells within the lung parenchyma. These lesions contain both TSC2-/- LAM cells, and wild type fibroblasts. We investigated the effect of TSC2-/- cells on the transcriptional profile of both normal fibroblasts and fibroblasts isolated from LAM patient tissue (LAFs) using Affymetrix Gene Chip arrays, to identify upregulated pathways which could contribute to LAM pathology. We used Affymetrix microarrays to detail the changes in the global profile of gene expression in fibroblasts following co-culture with 621-101 cells, and identified upregulation of several chemokines, indicating that the fibroblasts had adopted an inflammatory phenotype.
Project description:mTORC1 is inhibited by rapamycin or Torin1 in lymphangioleiomyomatosis (LAM) 621-101 (TSC2-deficient) cells, in which mTORC1 activity is elevated, and we compared these effects to inhibition of S6K and its target SRPK2.
Project description:Human retinal organoids have been invaluable in vitro models of retinal development and disease. To investigate whether cell states and gene expression changes observed in human fetal cone development are accurately replicated in organoids, we produced H9 hESC-derived retinal organoids using two published methods (Kuwahara et al. 2015, Nat Comm 6, 6286, https://doi.org/10.1038/ncomms7286; Zhong et al. 2014, Nat Comm 5, 4047, https://doi.org/10.1038/ncomms5047). We then FACS-enriched cone and rod precursors and retinal progenitor cells every two weeks from 55-140 DIC, as well as at 225 DIC, and performed deep, full-length scRNA-seq chemistry. These data were used to identify maturation differences related to the organoid production method, identify aberrant cell populations or gene expression timing relative to human fetal retina, and define cone and rod photoreceptor trajectories in the retinal organoid setting.
Project description:Rationale: Increased matrix metalloproteinase (MMP) activity has been implicated in the pathogenesis of lymphangioleiomyomatosis (LAM). Objectives: To investigate how TSC1 or TSC2 deficiency alters MMP expression and regulation. Methods: We studied immortalized cells that lack TSC2 derived from an angiomyolipoma (AML) of a LAM patient, and a TSC2 add back derivative; and murine embryonic fibroblast cells that lack Tsc1 or Tsc2 and respective controls. Global gene expression analysis was carried out in the AML and derivative cell lines. MMP levels in the conditioned media from these cells were analyzed by zymography and ELISA. Measurements and Main Results: We found increased MMP-2 expression in cells lacking TSC1/TSC2 compared to their respective controls by zymography. MMP-2 overproduction by these cells was not affected by rapamycin treatment. Gene expression analysis confirmed increased MMP-2 gene expression that was not affected by rapamycin. Furthermore, multiple other genes were found to be over-expressed in rapamycin-treated TSC2-deficient cells compared to TSC2+ cells. Conclusions: We conclude that TSC1/TSC2 deficiency leads to MMP-2 overproduction that is rapamycin insensitive, and that several genes exhibit similar patterns suggesting TSC1/TSC2 dependent but mTOR independent pathways may be involved in the pathogenesis of LAM.
Project description:Tuberous Sclerosis Complex (TSC) is caused by germline TSC1 or TSC2 mutations, leading to hyperactivation of mechanistic target of rapamycin complex 1 (mTORC1) and tumors in multiple organs including the brain, heart, lung (lymphangioleiomyomatosis), and kidney (angiomyolipoma and renal cell carcinoma). Previously, we found that TFEB is constitutively active in models of TSC. To determine the impact of TFEB in vivo, we generated two novel mouse models of TSC, resulting in premature death, in which kidney pathology was the primary phenotype. RNA sequencing revealed that lysosomal and proteasomal gene pathways were the most highly upregulated in the TSC2-deficient kidneys. Knockout of TFEB rescued both kidney pathology and overall survival in both models, indicating that TFEB is the primary driver of renal disease in TSC. Importantly, mTORC1 activity, which was elevated in the TSC2 knockout kidneys, was normalized by TFEB knockout. Knockdown of Rheb or treatment of TSC2-deficient cells with Rapamycin paradoxically increases TFEB phosphorylation at the mTORC1-site (S211) and relocalizes TFEB from the nucleus to the cytoplasm via a Rag-dependent mechanism. Accordingly, treatment of TSC2 knockout mice with Rapamycin normalized lysosomal gene expression, similar to TFEB knockout, suggesting that the beneficial effects of Rapamycin in TSC are TFEB-dependent. These results change the view of the mechanisms leading to mTORC1 hyperactivation in TSC and may lead to novel therapeutic avenues for the treatment of TSC.
Project description:We report results of RNA sequencing analysis of serum starved UMB1949 renal angiomyolipoma (AML) cells comparatively treated for 24hrs at 37C and 5% CO2 with rapamycin (0.05uM and 1uM) and tyrosine kinase inhibitor (TKI) imatinib (1uM and 10uM). Experiments were done in triplicates per drug concentration. This study was performed to investigate the biological mechanisms underlying superior cytocidal capabilities of the TKI over FDA-approved rapamycin by inhibiting receptor tyrosine kinases on mesenchymal tumorigenic cells in Tuberous Sclerosis and Lymphangioleiomyomatosis (LAM) diseases. Total RNA isolates (RIN>8.0) were sequenced on an illumina Novaseq6000 platform at 100 base pairs to a depth of 30 milllion single end reads. Real-Time Analysis was used for base callling converted to fastq format with bcl2fastq2. Pseudoalignment of RNA-seq reads to a kallisto index was created from human reference genome NCBI/GRCh38.p13. Reads were also mapped to this reference genome using STAR (2.5.2b) and featureCounts (v1.5.0-p3) for results comparison. Differential expression was resolved using Sleuth and DESeq2 in R and signaling pathway analysis was performed using Ingenuity Pathway Analysis (IPA). Results reveal differential inactivation of the GPVI pathway and key cell survival and viability genes only in renal AML cells treated with imatinib conentrations.We further determined that this TKI differentially induced ER calcium efflux that disrupted mitochondrial permeability leading to increased cytosolic cytochrome C, caspase 3 activation and apoptosis.