Expression data in PAX6 depleted corneal epithelial cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE198924 | GEO | 2022/07/27
REPOSITORIES: GEO
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Project description:Lineage-specific transcription factors (TFs) play key roles in maintaining the unique properties of cells, but the molecular mechanism that regulates the homeostasis of human corneal epithelial cells (CECs) is still poorly understood. We aimed to modulate KLF4 and PAX6 in human CECs using gene knockout system to clarify the regulatory network of KLF4, and then elucidate how KLF4 regulates the transcriptional genes of human CECs compared with PAX6. We performed a functional analysis of KLF4 via gene knockout using a lentivirus vector that carries both Cas9 and guide RNAs. We designed guide RNAs targeted for KLF4 and PAX6, and created KLF4-, PAX6-, and both KLF4- and PAX6-depleted CECs (KLF4-KO, PAX6-KO, and DKO, respectively). An empty vector was used as a control. The morphology of KLF4-KO CECs displayed an epithelial-mesenchymal transition (EMT)-like change, including upregulation of mesenchymal genes and downregulation of epithelial genes, as well as downregulation of keratin (KRT) 3 and KRT12. Global analyses using NGS revealed that the downregulated genes in KLF4-KO CECs were enriched in more widely keratin-related genes than PAX6-KO CECs. DKO cells showed disruption of the epithelial barrier due to downregulation of epithelial genes and showed more increase of KRT1 and KRT10 than PAX6-KO and KLF4-KO CECs, respectively. In conclusion, KLF4 modulates keratin-related genes as well as EMT-related genes and, together with PAX6, co-regulates the human CEC identity.
2022-07-27 | GSE198977 | GEO
Project description:The human genome encodes two proteins closely related to transketolase (TKT), transketolase-like protein 1 and 2 (TKTL1 and TKTL2). TKTL1 shares 61% amino acid sequence identity with TKT. More and more evidence suggests a role for TKTL1 in proliferation or cell cycle regulation. Indeed, TKTL1 is overexpressed in various cancers and is correlated with poor prognosis in colon, urothelial, gastric, and lung cancers as well as in ocular adnexa carcinomas, rectum carcinomas, and laryngeal squamous cell carcinomas. Increased TKTL1 levels also correlate with esophageal squamous cell carcinoma metastasis and increased resistance against cisplatin chemotherapy in nasopharyngeal carcinomas. Moreover, TKTL1 overexpression promotes cell proliferation and enhanced tumor growth; in contrast, TKTL1 down regulation attenuates the proliferation of various types of cancer cells. However, the cellular functions and regulatory machineries of TKTL1 are still largely unknown. Through this proteomic analysis, we defined the protein-protein interaction map for the TKTL1 and uncovered the novel regulating function of TKTL1 in regulating TKT transketolase activity.
2019-11-12 | PXD013309 | Pride
Project description:Considerable interest has been generated for the development through cell-tissue engineering of suitable corneal endothelial graft alternatives, which can potentially alleviate the shortage of corneal transplant material. The advent of less invasive suture-less key-hole surgery options such as DescemetM-bM-^@M-^Ys Stripping Endothelial Keratoplasty (DSEK) and DescemetM-bM-^@M-^Ys Membrane Endothelial Keratoplasty (DMEK), which involve transplantation of solely the endothelial layer instead of full thickness cornea, provide further impetus for the development of alternative endothelial grafts for clinical applications. A major challenge for this endeavor is the lack of specific markers for this cell type. To identify genes that reliably mark corneal endothelial cells (CECs) in vivo and in vitro, we performed RNA-sequencing on freshly isolated human CECs (from both young and old donors), CEC cultures, and corneal stroma. Gene expression of these corneal cell types were also compared to that of other human tissue types. Based on high throughput comparative gene expression analysis, we identified a panel of markers that are: i) highly expressed in CECs from both young donors and old donors; ii) expressed in CECs in vivo and in vitro; and iii) not expressed in corneal stroma keratocytes and the activated corneal stroma fibroblasts. These were SLC4A11, COL8A2 and CYYR1. The use of this panel of genes in combination reliably ascertains the identity of the CEC cell type. A total of 20 donor corneas consisting of 10 single donor corneas and 5 paired donor corneas were used in this study. Donor age ranged from 19 - 76. This RNA-seq study included 15 pooled corneas (5 each) used form CEC old, CEC young and stroma samples.
2013-05-23 | E-GEOD-44064 | biostudies-arrayexpress
Project description:Background: The underlying mechanism as well as diagnostic molecular biomarkers for corneal endothelial cell dysfunction (CECD) remain elusive. This study aimed to elucidate molecular mechanism of CECD via proteomic and transcriptomic approaches and identify novel biomarkers in aqueous humor (AH) directly associated with changes in CECs. Methods: We carried out an in-depth proteomic analysis of patient-derived AH as well as transcriptomic analysis of CECs from the same patients with bullous keratopathy (BK) resulting from CECD. Selected candidate proteins were further verified by individual data-independent acquisition and immunoassay using further AH samples from CECD patients. Findings: Using integrated multi-omics analysis, systematic alterations of proteins in AH and mRNA in CECs were mainly associated with the immune mechanism during BK development. Through individual proteomic verification via DIA analysis, three AH proteins were determined as potential predictive markers including TIMP1, FCGBP, and ANGPTL7. Finally, we confirmed these biomarkers using immunoassay in individual AH samples of CECD. Interpretation: Our multi-omics analysis may provide valuable insights into the disease-specific process as well as possibility of biofluid biomarkers in CECD. Further investigation with large scale is required to determine whether AH biomarkers are useful as liquid biopsy targets for the assessment of CEC function
2024-01-26 | PXD023772 | Pride
Project description:Purpose: We find that Wnt7a-PAX6 axis determine corneal epithelial cell fate. To obtain global evidence for successful cell fate conversion, we performed gene expression profiling by RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation. Methods: Under 3-D culture condition, limbal stem cell (LSCs) can be differentiated to Cornea epithelial cells (CECs), and skin epithelial stem cells (SESCs) can be differentiated to skin epithelial cells (SECs). Total RNA was isolated from CECs, SECs, and LSCs after knocking down PAX6 (3-D shPAX6 LSCs) and on SESCs transduced with PAX6 (3-D PAX6+ SESCs) upon 3-D differentiation. Libraries were prepared following published standard protocol (Fox-Walsh K et al., 2011, genomics, 266-71). mRNA profiles were generated by deep sequencing, in duplicate, using Illumina HiSeq 2000. Results: Following optimized decoding and mapping workfollow, we mapped about 5 million sequence reads to the human genome and identified more than 23659 transcripts per sample. Conclusions: Hierarchical clustering analysis of differentially expressed gene signatures revealed that the gene expression pattern of SESCs with PAX6 transduction was strikingly similar to that of CECs, whereas the profile of LSCs with PAX6 knockdown was highly related to that in SECs upon differentiation. These data therefore provided global evidence for a decisive role of the WNT7A/PAX6 axis in cell fate conversion from SESCs to CECs. RNA-seq on CECs, SECs, and LSCs after knocking down PAX6 and on SESCs transduced with PAX6 upon 3-D differentiation, using Illumina HiSeq 2000
2014-07-11 | E-GEOD-54322 | biostudies-arrayexpress
Project description:We examined the RNA expression profiles of whole skin from newborn CRISPR-edited mice that have the 923 enhancer located within the Epidermial Differentiation Complex deleted. We examined two independent deletion lines, 923del and 923large, both homozygous mutants and heterozygotes, as well as wild type mice and involucrin Ivl knockout mice. We find that Ivl, the gene most proximal to 923, is downregulated upon the loss of the enhancer in both independent deletions, and 4 additional genes are also downregulated in both deletion lines, a secondary effect of the loss of Ivl, as concluded by comparing to the Ivl KO mice. We thus conclude that Ivl is the primary target gene of the 923 enhancer and establish a 923-Ivl regulatory module.
2020-10-30 | GSE158869 | GEO
Project description:PURPOSE. Limbal stem/progenitor cells (LSC) continuously proliferate and differentiate to replenish the corneal epithelium and play a vital role in corneal function and normal vision. Previous study revealed that Paired box 6 (PAX6) is a master transcription factor involved in determining the fate of corneal epithelial cells (CEC). However, the molecular events downstream of PAX6 remain largely unknown. In this study, we aimed to clarify the regulation network of PAX6 in driving CEC differentiation. METHODS. An air-liquid culture system was used to differentiate LSC into mature CEC. Specific targeting PAX6 short-hairpin shRNAs were used to knock down PAX6 in LSC. RNA-seq was used to analyze shPAX6-transfected CEC and CEC differentiation-associated genes to identify the potential downstream targets of PAX6. RNA-seq analysis, quantitative real-time PCR, and immunofluorescence staining were performed to clarify the function of WNK lysine deficient protein kinase 2 (WNK2), a downstream target of PAX6, and its relationship with corneal diseases. RESULTS. WNK2 expression increased during CEC differentiation and decreased upon PAX6 depletion. The distribution of WNK2 was specifically limited to the central corneal epithelium and suprabasal layer of the limbus. Knockdown of WNK2 impaired the expression of CEC-specific markers (KRT12, ALDH3A1, and CLU), disrupted the corneal differentiation process, and activated the terms of keratinization, inflammation, and cell proliferation, consistent with PAX6-depleted CEC and published microbial keratitis. Thus, aberrant expression of WNK2 was linked to corneal ulcers. CONCLUSIONS. As a downstream target of PAX6, WNK2 plays an essential role in corneal epithelial cell differentiation and maintenance of corneal homeostasis.
2024-10-30 | GSE272034 | GEO
Project description:Corneal endothelium is composed of a monolayer of corneal endothelial cells (CECs) in the inner layer of cornea, which is essential for maintaining corneal transparency. In order to better characterize CECs in different developmental stages, we profiled mRNA transcriptomes in human fetal and adult corneal endothelium with the goal to identify novel molecular markers in these cells. By comparing CECs with 12 other types of tissues, we identified 245 and 284 signature genes that are highly expressed in fetal and adult CECs, respectively. Functionally, these genes are characteristic of CECs, involving in cell adhesion, proteoglycan and sulfur metabolic process. Importantly, several of these genes are disease target genes in hereditary corneal dystrophies, consistent with their functional significance in CEC physiology. By comparing fetal and adult CECs, we also identified stage-specific markers associated with CEC maturation, such as the activation of the Wnt pathway genes in fetal, but not in adult CECs. Lastly, by immunohistochemistry of ocular tissues, we further confirmed the unique protein expression patterns for Wnt5a, S100A4, S100A6, and IER3, the four novel markers for either fetal or adult CECs. The identification of a new panel of molecular markers for fetal and mature CECs would be very useful for characterizing and quality controlling CECs through ex vivo expansion or stem cell differentiation for cell replacement therapy. mRNA profile between adult and fetal CECs by high-throughput sequencing
2013-01-08 | E-GEOD-41616 | biostudies-arrayexpress
Project description:Non-oxidative pentose phosphate pathway (PPP) is a crucial gatekeeper of glucose catabolism in metabolic tissues. However, its role in regulatory T cells (Tregs) remains unknown. Here we report deleting transketolase (TKT), an indispensable enzyme of non-oxidative PPP, in Tregs caused a fatal auto-immune disease in mice. TKT deletion impaired suppressive capability without disturbing Treg cell number. Mechanistically, TKT deficiency caused Treg metabolic remodeling with decreased glucose catabolism, activated fatty acid and amino acid catabolism and uncontrolled oxidative phosphorylation. Moreover, excessive ammonia from deregulated amino acid catabolism impaired mitochondrial fitness while reduced α-ketoglutarate/succinate ratio led to DNA hypermethylation, limiting functional gene expression and suppressive activity of TKT-deficient Tregs. Therefore, our study identifies non-oxidative PPP as a new pathway for controlling Treg function.
2022-03-31 | GSE172047 | GEO
Project description:Non-oxidative pentose phosphate pathway (PPP) is a crucial gatekeeper of glucose catabolism in metabolic tissues. However, its role in regulatory T cells (Tregs) remains unknown. Here we report deleting transketolase (TKT), an indispensable enzyme of non-oxidative PPP, in Tregs caused a fatal auto-immune disease in mice. TKT deletion impaired suppressive capability without disturbing Treg cell number. Mechanistically, TKT deficiency caused Treg metabolic remodeling with decreased glucose catabolism, activated fatty acid and amino acid catabolism and uncontrolled oxidative phosphorylation. Moreover, excessive ammonia from deregulated amino acid catabolism impaired mitochondrial fitness while reduced α-ketoglutarate/succinate ratio led to DNA hypermethylation, limiting functional gene expression and suppressive activity of TKT-deficient Tregs. Therefore, our study identifies non-oxidative PPP as a new pathway for controlling Treg function.
2022-03-31 | GSE171675 | GEO