Chimeric AGO2-miR-eCLIP analysis of LIMD1 deficient (CRISPR-Cas9 edited) human SAEC cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE304955 | GEO | 2026/06/23
REPOSITORIES: GEO
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Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
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Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
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Project description:Growing demand in RNA-targeted therapies and promise of miRNA-based drugs creates a need for tools that can accurately identify and quantify miRNA:target interactions at scale. The experimental capture of miRNA:mRNA interactions by ligation into chimeric RNA fragments provides a direct read out of miRNA targets by enabling profiling of miRNA targets with high-throughput sequencing. However, integration of chimeric CLIP-seq into wide practical use has been limited because the inefficiency of the miRNA:mRNA ligation step (resulting in a low rate of chimeric reads in final libraries) combined with the technical complexity of the method makes it challenging to apply to miRNAs of interest at scale. Here we describe chimeric eCLIP, in which we integrate a chimeric ligation step into AGO2 eCLIP to enable chimeric read recovery, and show that removal of the cumbersome polyacrylamide gel and nitrocellulose membrane transfer step common to CLIP techniques can be omitted for chimeric AGO2 eCLIP to create a simplified high throughput version of the assay that maintains high signal-to-noise. With the increased yield of recovered miRNA:mRNA interactions in no-gel chimeric eCLIP, we show that simple enrichment steps using either PCR or on-bead probe capture can be added to chimeric eCLIP in order to target and enrich libraries for chimeric reads specific to one or more miRNAs of interest in both cell lines and tissue samples, resulting in 30- to 175-fold increases in recovery of chimeric reads for miRNAs of interest. We further show that the same probe-capture approach can be used to recover miRNA interactions for a targeted gene of interest, revealing both distinct miRNA targeting as well as co-targeted by several miRNAs from the same seed family. RNA-seq analysis of gene expression following miRNA overexpression confirmed miRNA-mediated repression of chimeric eCLIP identified targets, and indicated that chimeric eCLIP can provide additional sensitivity to detect regulated targets among genes that either contain or lack computationally predicted miRNA target sites. Thus, we believe that chimeric eCLIP will be a useful tool for quantitative profiling of miRNA targets in varied sample types at scale, and revealing a deeper picture for regulatory networks for miRNAs of biological interest.
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Project description:Increasing evidence suggests that global downregulation of miRNA expression is a hallmark of human cancer, potentially due to defects in the miRNA processing machinery. In this study, we found that the protein expression of Argonaute 2 (AGO2), a key regulator of miRNA processing, was downregulated in colorectal cancer (CRC) tissues, which was also consistent with the findings of the Clinical Proteomic Tumor Analysis Consortium (CPTAC). Furthermore, the correlation between the levels of AGO2 and epithelial-mesenchymal transition (EMT) markers (E-cadherin and vimentin) indicated that reduced levels of AGO2 promoted EMT in CRC. Low expression of AGO2 was an indicator of a poor prognosis among CRC patients. Knockdown of AGO2 in CRC cells promoted migration, invasion and metastasis formation in vitro and in vivo but had no influence on proliferation. To provide detailed insight into the regulatory roles of AGO2, we performed integrated transcriptomic, quantitative proteomic and microRNA sequencing (miRNA-seq) analyses of AGO2 knockdown cells and the corresponding wild-type cells and identified neuropilin 1 (NRP1) as a new substrate of AGO2 via miR-185-3p. Our data provided evidence that knockdown of AGO2 resulted in a reduction of miR-185-3p expression, leading to the upregulation of the expression of NRP1, which is a direct target of miR-185-3p, and elevated CRC cell metastatic capacity. Inhibition of NRP1 or treatment with a miR-185-3p mimic successfully rescued the phenotypes of impaired AGO2, which suggested that therapeutically targeting the AGO2/miR-185-3p/NRP1 axis may be a potential treatment approach for CRC.
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