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Direct detection of circulating microRNA-122 using dynamic chemical labelling with single molecule detection overcomes stability and isomiR challenges for biomarker qualification

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE140833 | GEO | 2019/11/23

REPOSITORIES: GEO

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Project description:Background/Aim: We investigated alterations in the expression of serum exosomal miRNAs with the progression of liver fibrosis and evaluated their clinical applicability as biomarkers. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Methods: This study prospectively enrolled 71 patients who underwent liver biopsy at an academic hospital in Korea. Exosomes were extracted from serum samples, followed by next-generation sequencing (NGS) of miRNAs and targeted real-time quantitative polymerase chain reaction. A model was derived to discriminate advanced fibrosis based on miRNA levels and the performance of this model was evaluated. Validation of the effect of miRNA on liver fibrosis in vitro was followed. Results: NGS data revealed that exosomal miR-660-5p, miR-125a-5p, and miR-122 expression were changed significantly with the progression of liver fibrosis, of which miR-122 exhibited high read counts enough to be used as a biomarker. The level of exosomal miR-122 decreased as the pathologic fibrosis grade progressed and patients with biopsy-proven advanced fibrosis had significantly lower levels of exosomal miR-122 (P < 0.001) than those without advanced fibrosis. Exosomal miR-122 exhibited a fair performance in discriminating advanced fibrosis especially in combination with fibrosis-4 score and transient elastography. In a subgroup of patients with a non-viral etiology of liver disease, the performance of exosomal miR-122 as a biomarker was greatly improved. Inhibition of miR-122 expression increased the proliferation of the human hepatic stellate cell line, LX-2, and upregulated the expression of various fibrosis related proteins. Conclusion: Exosomal miR-122 may serve as a useful non-invasive biomarker for liver fibrosis, especially in patients with non-viral etiologies of chronic liver disease.

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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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