Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification
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PROVIDER: PRJNA591119 | ENA |
REPOSITORIES: ENA
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Project description:Circulating microRNAs are biomarkers reported to be stable and translational acrossspecies. miR-122 (miR-122-5p) is a hepatocyte-specific microRNA biomarker for drug-induced liver injury (DILI). Our objective was to develop an extraction-free andamplification-free detection method for measuring miR-122 that has translationalutility in context of DILI. We developed a single molecule dynamic chemical labelling(DCL) assay based on miR-122 hybridization to an abasic peptide nucleic acid probethat contained a reactive amine instead of a nucleotide at a specific position in thesequence. The single molecule DCL assay specifically measured miR-122 directly from10 μL of serum or plasma without any extraction steps, with a fit-for-purpose limit ofdetection of 1.32 pM. In 192 human serum samples, DCL accurately identified patientsat risk of DILI (area under ROC curve 0.98 (95%CI 0.96-1), P<0.0001). The miR-122assay also quantified liver injury in rats and dogs. When DCL beads were added toserum, the miR-122 signal was stabilised (no loss of signal after 14 days at roomtemperature). By contrast, there was substantial degradation of miR-122 in the absenceof beads (≈60% lost in 1 day). RNA sequencing demonstrated the presence of multiplemiR-122 isomiRs with DILI that were at low concentration or not present in healthypatient serum. Sample degradation over time produced more isomiRs, particularlyrapidly with DILI. PCR was inaccurate when analysing miR-122 isomiRs, whereas theDCL assay demonstrated accurate quantification. In summary, the DCL assay canaccurately measure miR-122 directly from serum and plasma to diagnose liver injury inhumans and other species, and can overcome important microRNA biomarker analyticaland biological challenges.
2019-11-23 | GSE140833 | GEO
Project description:Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5’UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect could be relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and number of sites. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.
2015-01-06 | GSE64679 | GEO
Project description:Detection of transcriptome changes associated with miR-122 inactivation in mouse liver at two Zeitgeber time points, ZT0 and ZT12.
2009-06-03 | E-TABM-692 | biostudies-arrayexpress
Project description:The liver-specific microRNA, miR-122, is an essential host factor for replication of hepatitis C virus (HCV), an important infectious cause of chronic liver disease and hepatocellular carcinoma. miR-122 stabilizes the positive-strand HCV RNA genome and promotes viral RNA synthesis by binding two closely spaced sites (S1 and S2) near the 5’ end of the genome in association with Ago2. Ago2 is essential for both host factor activities, but whether other host proteins are involved is unknown. Using a quantitative proteomics approach, we identified TNRC6A (GW182) and its paralogs (TNRC6B and TNRC6C), as functionally important components of the miR-122/Ago2 host factor complex binding HCV RNA. Depletion of any two TNRC6 proteins reduced HCV replication in Huh-7.5 cells,but did not reduce viral RNA stability or translational activity, but rather dampened miR-122 stimulation of viral RNA synthesis. However, TNRC6 depletion had no effect on replication of HCV in which S2 was mutated so that miR-122 binds only S1, whereas it significantly enhanced replication when S1 was mutated and only S2 bound by miR-122. Consistent with this, we found that TNRC6 proteins preferentially associate with the S1 site, and that the association of Ago2 with S2 is increased in TNRC6-depleted cells. Collectively, these data suggest a model in which TNRC6 proteins, which are known to interact with Ago2, preferentially direct the miR-122/Ago2 complex to S1 while restricting its association with S2, thereby fine tuning the spatial organization of miR-122/Ago2 complexes bound to the viral RNA.
2019-04-24 | PXD013054 | Pride
Project description:Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5’UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect could be relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and number of sites. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV. AGO HITS-CLIP libraries were generated from single cell clones of miR-122 deleted Huh7.5 cells using CRISPR (KO), unedited controls (WT), or cells transfected with GFP instead of CRISPR. Libraries were generated with a 4nt index read, a common priming sequence, followed by a 5nt degenerate barcode terminiating in a G. Files have been demultiplexed such that the 5nt degenerate barcode has been appended as the first 5 nucleotides of the read.
2015-01-06 | E-GEOD-64679 | biostudies-arrayexpress
Project description:In the recent past, the impact of isomiR expression changes in cancer has gained increasing interest. Here, we stably overexpress pre-miR-1307, a pre-miRNA which is frequently upregulated in cancer, and assess the effect on isomiR expression. While we see robust and significant upregulation of the canonical form miR-1307-3p|0 and its abundant 5'isomiR miR-1307-3p|1, as well as of the canonical miR from the 5p arm (miR-1307-5p|0), the overexpression of pre-miR-1307 did not lead to global alterations in isomiR expression patterns. These data validate the suitability of our model cell line for the analysis of the effects of joint upregulation of miR-1307-3p|0, -3p|1 and -5p|0.
2024-01-11 | GSE227354 | GEO
Project description:Hepatic injury is often accompanied by pulmonary inflammation and tissue damage, but the underneath mechanism is not fully elucidated. Here we identify hepatic miR-122 as a culprit of pulmonary inflammation induced by various liver injuries. Analyses of acute and chronic liver injury mouse models confirm that liver dysfunction can cause pulmonary inflammation and tissue damage. Injured livers release large amounts of miR-122 in a microvesicle-independent manner into the circulation compared to normal livers. Circulating miR-122 is then preferentially transported to mouse lungs and taken up by alveolar macrophages, in which it binds toll-like receptor 7 (TLR7) and activates inflammatory responses. Depleting plasma miR-122 largely abolishes liver injury-induced pulmonary inflammation and tissue damage. Furthermore, alveolar macrophage activation by miR-122 is blocked by mutating the TLR7-binding UG-rich sequence on miR-122 or knocking out macrophage TLR7. Our findings reveal a novel causative role of hepatic miR-122 in liver injury-induced pulmonary dysfunction.
2018-12-27 | GSE124360 | GEO
Project description:Hepatitis C virus uniquely requires the liver specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (Ago) during HCV infection showed robust Ago binding on the HCV 5’UTR, at known and predicted miR-122 sites. On the human transcriptome, we observed reduced Ago binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect could be relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and number of sites. We describe a quantitative mathematical model of HCV induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.
2015-01-06 | GSE64677 | GEO
Project description:Identification of 5'isomiR in HCC patients.
| PRJNA730731 | ENA