Скачать книгу

the intracellular small RNA content (~55% of the reads), miRNAs were only the fourth most populated of the EV-derived small RNAs, at around 10% of the total reads. Later studies have both supported and contradicted these findings. For example, Sork et al. (2018) also described different patterns of intracellular small, ribosomal, and other RNAs versus those found in EVs. Of the total reads, small RNAs were a minority; they were noted in the five cell cultures assessed, ranging from ~ 2% to ~ 40% of the reads. However, 58%–83% of the small RNA EV reads were attributed to miRNAs, many of these reflecting those found in the parent cell, with some notable exceptions such as miR-451a. The disagreements between different studies could be due to cell types, methods, and the analyses used. Overall, from a biomarker perspective, the content of these EVs reflects the cellular state and can be specific to a cell type by partially reflecting the transcriptome (Sork et al. 2018; Srinivasan et al. 2019); yet the EVs differ in intracellular RNA content from the source cell (Guduric-Fuchs et al. 2012; Zhang et al. 2010). Therefore packaging these EVs is not merely representative of the RNA cellular milieu; it is rather an active process that may be influenced by cellular responses to stressors.

      Biological Interpretations Due to Mechanisms of Release

      The difference between a “leakage” miRNA biomarker and one that is non-randomly packaged into an EV as a putative communication signal, is an important distinction for development of miRNA biomarkers that are indicative of health effects. For example, mammalian miR-122 has been heavily investigated as a leakage biomarker for liver toxicity, in the context of both non-clinical and clinical species. miR-122 accounts for approximately 72% of the total liver miRNAs in mice (Lagos-Quintana et al. 2002) and is highly specific for human, mouse, and rat livers (Landgraf et al. 2007; Smith et al. 2016). In a landmark study by Wang et al. (2009), mice were treated with hepatotoxic levels of acetominophen (APAP) and miRNA microarrays were used to measure difference in the plasma. Forty-four miRNAs were significantly altered, miR-122 showing a 470-fold increase twenty-four hours after APAP overdose, and these alterations could be observed as soon as one hour after exposure. This APAP overdose or acute liver injury-associated miR-122 release was later confirmed in human patients (Starkey Lewis et al. 2011). The observation that decreased liver-based miRNAs with APAP hepatotoxicity correlated with increases in serum plasma suggested that these miRNAs were released as packaged contents or leaked during hepatocyte necrosis; the leakage theory was supported by Arroyo et al. (2011), who demonstrated that miR-122 was primarily associated with a RNP complex (Ago2) and was not membrane-bound as in exosomes. However, later studies provided clues that the mechanism of miR-122 release from liver was context-dependent, that is, dependent on factors such as the condition of the liver, the timing of the injury or perturbation, the half-life of a miRNA, and baseline variability. Subtoxic exposures in vitro result primarily in hepatocyte-derived release of exosome-bound miR-122 (Holman et al. 2016; Mosedale et al. 2018), and alcoholic and inflammatory liver disease was associated with exosome-bound miR-122—as opposed to drug-induced injury, which was found in the protein-bound fraction of plasma (Bala et al. 2012). Further, the protection of miRNA from RNases present in biofluids may preferentially enrich exosome and protein-bound miRNAs (Arroyo et al. 2011; Koberle et al. 2013; Li et al. 2012; Turchinovich et al. 2011).

      MicroRNAs as Bioindicators of Toxicity and Disease

      Much miRNA biomarker research has been focused on the early detection of drug-induced liver injury (DILI), a major roadblock in drug development. Alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, and total bilirubin are the currently approved DILI biomarkers in clinical practice (Robles-Diaz et al. 2016). These biomarkers are not specific to hepatotoxicity, as they increase in almost all abnormal liver conditions. Furthermore, they lack sensitivity, as they appear once liver damage has developed, and consequently have limited use for predicting potential liver injury at an early stage. For this reason, circulating miRNAs are being investigated for the monitoring of drug-induced tissue injury (tissue degeneration and necrosis) in cases where highly abundant tissue-specific miRNAs are passively released after cell injury. In incidences of DILI, studies have demonstrated that miR-122 presence in blood correlates tightly with pathological indications and clinical chemistries (i.e., ALT, AST) of liver

Скачать книгу