Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

ABSTRACT: TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase Z^L, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.

SUBMITTER: Nozaki T

PROVIDER: S-EPMC7859166 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

The heptamer sgRNA targeting the human OCT4 mRNA can upregulate the OCT4 expression.

Nozaki Tadasuke T Takahashi Masayuki M Ishikawa Tatsuya T Haino Arisa A Seki Mineaki M Kikuchi Hidetomo H Yuan Bo B Nashimoto Masayuki M

Biochemistry and biophysics reports 20210202

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase Z<sup>L</sup>, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human am ...[more]

PMID: 33553691

Similar Datasets

BAY11 enhances OCT4 synthetic mRNA expression in adult human skin cells.

Project description:INTRODUCTION:The OCT4 transcription factor is involved in many cellular processes, including development, reprogramming, maintaining pluripotency and differentiation. Synthetic OCT4 mRNA was recently used (in conjunction with other reprogramming factors) to generate human induced pluripotent stem cells. Here, we discovered that BAY 11-7082 (BAY11), at least partially through an NF-?B-inhibition based mechanism, could significantly increase the expression of OCT4 following transfection of synthetic mRNA (synRNA) into adult human skin cells. METHODS:We tested various chemical and molecular small molecules on their ability to suppress the innate immune response seen upon synthetic mRNA transfection. Three molecules - B18R, BX795, and BAY11 - were used in immunocytochemical and proliferation-based assays. We also utilized global transcriptional meta-analysis coupled with quantitative PCR to identify relative gene expression downstream of OCT4. RESULTS:We found that human skin cells cultured in the presence of BAY11 resulted in reproducible increased expression of OCT4 that did not inhibit normal cell proliferation. The increased levels of OCT4 resulted in significantly increased expression of genes downstream of OCT4, including the previously identified SPP1, DUSP4 and GADD45G, suggesting the expressed OCT4 was functional. We also discovered a novel OCT4 putative downstream target gene SLC16A9 which demonstrated significantly increased expression following elevation of OCT4 levels. CONCLUSIONS:For the first time we have shown that small molecule-based stabilization of synthetic mRNA expression can be achieved with use of BAY11. This small molecule-based inhibition of innate immune responses and subsequent robust expression of transfected synthetic mRNAs may have multiple applications for future cell-based research and therapeutics.

| S-EPMC3706837 | biostudies-literature

C-MYC controlled TIP110 protein expression regulates OCT4 mRNA splicing in human embryonic stem cells.

Project description:We reported previously that HIV-1 the Tat-interacting protein of 110 kDa (TIP110; P110(NRB)/SART3/p110) is important in regulation of hematopoiesis, and in maintaining pluripotent factor (NANOG, OCT4, and SOX2) expression in and pluripotency of human embryonic stem cells (hESCs). Here we show that TIP110 expression in hESCs line H9 and embryonal carcinoma cell line NT-2 is regulated by C-MYC expression in ESCs through an E box present in the TIP110 promoter region. Through up- and down- modulation of expression, TIP110 induces OCT4 exon 1a inclusion and exon 1b skipping in our OCT4 minigene model. Thus, TIP110 expression in ESCs regulates alternative splicing of OCT4, an event required for pluripotency of ESCs.

| S-EPMC3578368 | biostudies-literature

Synthesis of an arrayed sgRNA library targeting the human genome.

Project description:Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested.

| S-EPMC4597219 | biostudies-literature

Screening of a heptamer-type sgRNA library for therapeutic agents against hematological malignancies.

Project description:tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5M-bM-^@M-2-half-tRNA or microRNA as sgRNA. In order to estimate a number of potential therapeutic heptamer-type sgRNAs for hematological malignancies, we constructed an sgRNA library composed of 156 heptamer-type sgRNAs, and examined how the sgRNAs affect viability of leukemia and myeloma cells. And we found that 20 of the 156 sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. Furthermore, we demonstrated that 4 of the 20 effective sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. DNA microarray analysis for changes in an mRNA profile by these four heptamer-type sgRNAs suggested at least one candidate target mRNA that contains a potential tRNase ZL target site for each sgRNA. Changes in gene expression in HL60 cells were measured after 18-hour incubation in the absence or presence of one of five different heptamer-type sgRNAs. *Heptamer sequences requested but not provided by submitter

2014-06-30 | E-GEOD-49578 | biostudies-arrayexpress

Screening of a heptamer-type sgRNA library for therapeutic agents against hematological malignancies.

Project description:tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing) is an RNA-mediated gene expression control technology that has therapeutic potential. This technology is based on the properties of tRNase ZL that it can cleave any target RNA at any desired site under the direction of an appropriate artificial small guide RNA (sgRNA) and that cytosolic tRNase ZL can modulate gene expression by cleaving mRNA under the direction of cellular 5′-half-tRNA or microRNA as sgRNA. In order to estimate a number of potential therapeutic heptamer-type sgRNAs for hematological malignancies, we constructed an sgRNA library composed of 156 heptamer-type sgRNAs, and examined how the sgRNAs affect viability of leukemia and myeloma cells. And we found that 20 of the 156 sgRNAs can efficiently induce apoptosis in at least one of the cancer cell lines. Furthermore, we demonstrated that 4 of the 20 effective sgRNAs can reduce growth rates of HL60 cells in mouse xenograft models. DNA microarray analysis for changes in an mRNA profile by these four heptamer-type sgRNAs suggested at least one candidate target mRNA that contains a potential tRNase ZL target site for each sgRNA.

2014-06-30 | GSE49578 | GEO

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Project description:Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

| S-EPMC6023838 | biostudies-literature

BTK mutations selectively regulate BTK expression and upregulate monocyte XBP1 mRNA in XLA patients.

Project description:Mutations in the Bruton agammaglobulinemia tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA). Unfolded or misfolded proteins can trigger stress pathways in the endoplasmic reticulum (ER), known as unfolded protein response (UPR). The aim was to clarify the involvement of UPR in XLA pathophysiology. By reverse transcription-quantitative PCR, we evaluated the expression of BTK and 12 UPR-related genes in eight patients. Moreover, we assessed the BTK protein expression and pattern in the patients' monocytes by flow cytometry and fluorescence immunocytochemistry. We found a reduced BTK expression in patients with stop codon mutations (P?<?0.02). However, missense mutations did not affect BTK expression. Flow cytometry showed a reduction of BTK in patients which was corroborated by an absent or nonfunctional protein synthesis revealed by immunocytochemistry. In contrast with the other UPR-related genes, X-box binding protein 1 (XBP1) was markedly upregulated in the patients (P?<?0.01), suggesting Toll-like receptor (TLR) activation since BTK directly interacts with TLRs as a negative regulator and XBP1 can be activated in direct response to TLR ligation. Different BTK mutations can be identified by the BTK expression. Inasmuch as UPR-related genes were downregulated or unaltered in patients, we speculate the involvement of the TLRs-XBP1 axis in the XLA pathophysiology. Such data could be the basis for further studies of this novel pathomechanism concerning XLA.

| S-EPMC4578518 | biostudies-literature

Human transcription factor YY1 could upregulate the HIV-1 gene expression.

Project description:Gene expression in HIV-1 is regulated by the promoters in 5' long-terminal repeat (LTR) element, which contain multiple DNA regulatory elements that serve as binding sites for cellular transcription factors. YY1 could repress HIV-1 gene expression and latent infection. Here, however, we observed that virus production can be increased by YY1 over-expression and decreased under YY1 depleted condition by siRNA treatment. To identify functional domain(s) of YY1 activation, we constructed a number of YY1 truncated mutants. Our data show that full-length YY1 enhances the viral transcription both through U3 and U3RU5 promoters. Moreover, the C-terminal region (296-414 residues) of YY1 is responsible for the transcriptional upregulation, which could be enhanced further in the presence of the viral Tat protein. The central domain of YY1 (155-295 residues) does not affect LTR activity but has a negative effect on HIV-1 gene expression. Taken together, our study shows that YY1 could act as a transcriptional activator in HIV-1 replication, at least in the early stages of infection. [BMB Reports 2020; 53(5): 248-253].

| S-EPMC7262509 | biostudies-literature

RUNX1 and RUNX2 upregulate Galectin-3 expression in human pituitary tumors.

Project description:Galectin-3 is expressed in a cell-type specific manner in human pituitary tumors and may have a role in pituitary tumor development. In this study, we hypothesized that Galectin-3 is regulated by RUNX proteins in pituitary tumors. Transcription factor prediction programs revealed several putative binding sites in the LGALS3 (Galectin-3 gene) promoter region. A human pituitary cell line HP75 was used as a model to study LGALS3 and RUNX interactions using Chromatin immunoprecipitation assay and electrophoresis mobility shift assay. Two binding sites for RUNX1 and one binding site for RUNX2 were identified in the LGALS3 promoter region. LGALS3 promoter was further cloned into a luciferase reporter, and the experiments showed that both RUNX1 and RUNX2 upregulated LGALS3. Knock-down of either RUNX1 or RUNX2 by siRNA resulted in a significant downregulation of Galectin-3 expression and decreased cell proliferation in the HP 75 cell line. Immunohistochemistry showed a close correlation between Galectin-3 expression and RUNX1/RUNX2 level in pituitary tumors. These results demonstrate a novel binding target for RUNX1 and RUNX2 proteins and suggest that Galectin-3 is regulated by RUNX1 and RUNX2 in human pituitary tumor cells by direct binding to the promoter region of LGALS3 and thus may contribute to pituitary tumor progression.

| S-EPMC2927870 | biostudies-literature

Human haematopoietic stem cells express Oct4 pseudogenes and lack the ability to initiate Oct4 promoter-driven gene expression.

Project description:The transcription factor Oct4 is well defined as a key regulator of embryonic stem (ES) cell pluripotency. In recent years, the role of Oct4 has purportedly extended to the self renewal and maintenance of multipotency in adult stem cell (ASC) populations. This profile has arisen mainly from reports utilising reverse transcription-polymerase chain reaction (RT-PCR) based methodologies and has since come under scrutiny following the discovery that many developmental genes have multiple pseudogenes associated with them. Six known pseudogenes exist for Oct4, all of which exhibit very high sequence homology (three >97%), and for this reason the generation of artefacts may have contributed to false identification of Oct4 in somatic cell populations. While ASC lack a molecular blueprint of transcription factors proposed to be involved with 'stemness' as described for ES cells, it is not unreasonable to assume that similar gene patterns may exist. The focus of this work was to corroborate reports that Oct4 is involved in the regulation of ASC self-renewal and differentiation, using a combination of methodologies to rule out pseudogene interference. Haematopoietic stem cells (HSC) derived from human umbilical cord blood (UCB) and various differentiated cell lines underwent RT-PCR, product sequencing and transfection studies using an Oct4 promoter-driven reporter. In summary, only the positive control expressed Oct4, with all other cell types expressing a variety of Oct4 pseudogenes. Somatic cells were incapable of utilising an exogenous Oct4 promoter construct, leading to the conclusion that Oct4 does not appear involved in the multipotency of human HSC from UCB.

| S-EPMC2853495 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data