Gene expression changes of CRISPR/Cas13d-mediated HoxB13 knockdown in 293FT cells and LNCaP95 mouse liver metastasis
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1100904 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR28704457_1.fastq.gz | Fastqsanger.gz | |||
| SRR28704457_2.fastq.gz | Fastqsanger.gz | |||
| SRR28704458_1.fastq.gz | Fastqsanger.gz | |||
| SRR28704458_2.fastq.gz | Fastqsanger.gz | |||
| SRR28704459_1.fastq.gz | Fastqsanger.gz |
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Project description:To examine the potential off-target effects by CRISPR/Cas13d-mediated HoxB13 knockdown in 293FT cells. To analyze differentially expressed genes upon CRISPR/Cas13d-mediated HoxB13 knockdown in LNCaP95 mouse liver metastasis.
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Project description:HOXB13 is a prostate-specific homeodomain transcription factor that is mainly known as an androgen receptor (AR) cofactor. Recent studies have revealed AR-independent roles of HOXB13 in recruiting HDAC3 to suppress lipogenic programs in prostate cancer (PCa). As such, HOXB13 down-regulation leads to lipid accumulation and tumor metastasis, the molecular mechanisms of which, however, are incompletely understood. Here, we demonstrate that p300/CBP co-occupies HOXB13/HDAC3-repressed lipogenic enhancers and is required for their activation and target gene expression upon HOXB13 loss. We found that HOXB13 is down-regulated in metastatic hormone-sensitive PCa compared to their matched primary prostate tumors. HOXB13 loss induces matrix metalloproteinases (MMPs), which mediate HOXB13-depleted cell motility. Critically, CCS1477, a pharmacological inhibitor of p300/CBP, abolished HOXB13-loss-induced lipid accumulation, MMP gene expression, cell motility in vitro, and tumor metastasis in vivo. Taken together, our results suggest HOXB13 as a significant metastasis suppressor and identify p300/CBP as critical therapeutic targets in HOXB13-low metastatic PCa.
2025-10-08 | GSE296237 | GEO
Project description:HOXB13 is a prostate-specific homeodomain transcription factor that is mainly known as an androgen receptor (AR) cofactor. Recent studies have revealed AR-independent roles of HOXB13 in recruiting HDAC3 to suppress lipogenic programs in prostate cancer (PCa). As such, HOXB13 down-regulation leads to lipid accumulation and tumor metastasis, the molecular mechanisms of which, however, are incompletely understood. Here, we demonstrate that p300/CBP co-occupies HOXB13/HDAC3-repressed lipogenic enhancers and is required for their activation and target gene expression upon HOXB13 loss. We found that HOXB13 is down-regulated in metastatic hormone-sensitive PCa compared to their matched primary prostate tumors. HOXB13 loss induces matrix metalloproteinases (MMPs), which mediate HOXB13-depleted cell motility. Critically, CCS1477, a pharmacological inhibitor of p300/CBP, abolished HOXB13-loss-induced lipid accumulation, MMP gene expression, cell motility in vitro, and tumor metastasis in vivo. Taken together, our results suggest HOXB13 as a significant metastasis suppressor and identify p300/CBP as critical therapeutic targets in HOXB13-low metastatic PCa.
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Project description:RNA-guided and RNA-targeting type IV-D CRISPR/Cas systems (CRISPR/Cas13d) have recently been identified and employed for efficient and specific RNA knockdown in mammalian and plant cells. Cas13d possesses dual RNase activities and is capable of processing CRISPR arrays and cleaving target RNAs in a protospacer flanking sequence (PFS)-independent manner. These properties make this system a promising tool for multiplex gene expression regulation in microbes. Herein, we aimed to establish a CRISPR/Cas13d-mediated RNA knockdown platform for bacterial chassis. CasRx, Cas13d from Ruminococcus flavefaciens XPD3002, was selected due to its high activity. However, CasRx was found to be highly toxic to both Escherichia coli and Corynebacterium glutamicum, especially when it cooperated with its guide and target RNAs. After employing a low copy number vector, a tightly controlled promoter, and a weakened ribosome binding site, we successfully constructed an inducible expression system for CasRx and applied it for repressing the expression of a green fluorescent protein (GFP) in E. coli. Despite our efforts to optimize inducer usage, guide RNA (gRNA) architecture and combination, and target gene expression level, the highest gene repression efficiency was 30-50% at the protein level and ∼70% at the mRNA level. The moderate RNA knockdown is possibly caused by the collateral cleavage activity toward bystander RNAs, which acts as a mechanism of type IV-D immunity and perturbs microbial metabolism. Further studies on cellular response to CRISPR/Cas13d and improvement in RNA knockdown efficiency are required prior to practical application of this system in microbes.
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Project description:We performed an experimental Cas13d-SARScov2 genome-wide screen to identify gRNAs that would allow Cas13d to degrade the viral RNA. We built mCherry reporter plasmids that express mCherry with a 3kb 3'UTR deriving from the SARScov2 genome. In total we designed 11 reporters covering the entire plus strand of the viral genome and 11 other reporters covering the entire minus strand. Each of the 22 mCherry reporter plasmids carries a U6 expression cassette containing a Cas13d gRNA that targets the 3'UTR of the mCherry reporter. Each reporter is represented by a pool of reporters each containing a different gRNA that targets mCherry 3'UTR for a total average of ~300 gRNA per 3'UTR. The entire pool of 22 reporters, each with a pool of ~300 different gRNAs constitutes a comprehensive set ~6,500 reporters (~ 6,500 different gRNAs) that allowed us to interrogate the entire SARScov2 plus and minus strand viral RNA for regions of vulnerability and targetability. In order to specifically interrogate Cas13d activity an remove the biases that would be introduced in the reporter expression by the presence of a large 3kb 3'UTR we used a case (presence of Cas13d) control (absence of Cas13d) design. Briefly, the ~6,500 reporters were lentiviral transduced in RKO cells, the cells were split in 2 populations, 1 population was transduced with Cas13d and the other serving as control did not. The population expressing Cas13d was FACS sorted in low mCherry (efficient gRNAs) and high mCherry (un-efficient gRNAs) in 2 biological replicates and the genomic DNA of these populations was extracted, gRNAs were PCR amplified and sequenced. For the population that did not express Cas13d, a low mCherry, one high mCherry and unsorted population were sequenced as control libraries.
2023-10-29 | E-MTAB-11111 | biostudies-arrayexpress
Project description:Circular RNAs (circRNAs) are widely expressed, but their functions remain largely unknown. To study circRNAs in a high-throughput manner, short hairpin RNA (shRNA) screens1 have recently been used to deplete circRNAs by targeting their unique back-splicing junction (BSJ) sites. Here, we report frequent discrepancies between shRNA-mediated circRNA knockdown efficiency and the corresponding biological effect, raising pressing concerns about the robustness of shRNA screening for circRNA functional characterization. To address this issue, we leveraged the CRISPR/Cas13d system2 for functional study of circRNAs by optimizing the strategy for designing single guide RNAs to deplete circRNAs. We then performed shRNA and CRISPR/Cas13d parallel screenings and demonstrated that shRNA-mediated circRNA screening yielded a high rate of false positive phenotypes. Furthermore, using a CRISPR/Cas13d screening library targeting over 2,500 human hepatocellular carcinomas (HCC) related circRNAs, we identified a group of circRNAs, whose inhibition increased the therapeutic efficacy of sorafenib, an approved HCC drug. Collectively, these data demonstrate that CRISPR/Cas13d system is an effective approach to study the function of circRNAs in a high-throughput manner.
2020-12-06 | GSE162720 | GEO
Project description:HOXB13 is a developmentally regulated transcription factor, co-expressed along with the Androgen receptor (AR) in a majority of PCs. Previous studies have indicated context dependent roles of HOXB13 in the functional regulation of AR and enrichment of HOXB13 motifs in the AR cistrome. Our studies showed that genetic or pharmacological blockade of HOXB13 sensitized mCRPCs to Enzalutamide (ENZ, Xtandi), an anti-androgen that is currently deployed to treat mCRPC patients. To identify HOXB13-mediated mechanism of castration-resistance ChIP-sequencing was performed with the HOXB13-K13ac, or IgG antibodies in the metastatic prostate cancer cell line, C4-2B in vehicle treated as well as Enzalutamide treated cell lines. We demonstrated for the first time that BRD4, epigenetically regulates HOXB13 gene expression in PCs. Consistently, JQ1 the prototype BET inhibitor suppresses HOXB13 expression and inhibit mCRPC growth. ChIP-sequence analysis reveals that HOXB13 recruitment to the chromatin is not completely abrogated in Enzalutamide treated cells identify a subset of genes that may have potential role in CRPC proliferation, anti-androgen resistance and metastasis.
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Project description:To identify potential cofactors of HOXB13 in suppressing lipogenic programs in prostate cancer cells, we performed tandem affinity purification followed by mass spectrometry analysis of WT and G84E HOXB13 expressed in LNCaP cells. Out of the HOXB13-enriched proteins are previously reported interactors such as AR and its cofactors FOXA1, GATA2, and NKX3. However, these interactions were not disrupted by G84E as compared to WT HOXB13. Interestingly, we found strong interactions of HOXB13 with HDAC1/3 and their corepressors NCoR1/2 and TBL1X. Notably, these interactions were drastically reduced by G84E mutation.
2023-03-11 | PXD030810 | Pride