Direct detection of endogenous MicroRNAs and their post-transcriptional modifications in cancer serum by capillary electrophoresis-mass spectrometry

Abstract

MicroRNA molecules (miRNAs) are a class of small, single-stranded, non-coding RNA molecules that regulate cellular messenger RNA and their corresponding proteins. Extracellular miRNAs circulate in the bloodstream inside exosomes or in complexes with proteins and lipoproteins. The miRNA sequences and their quantitative levels are used as unique signatures associated with cancer diagnosis and prognosis after anticancer treatment. MicroRNAs are modified through a series of processing events after transcription like 5'-end phosphorylation, 3'- end adenylation or uridylation, terminal nucleotide deletion. The problem is that existing bioanalytical methods such as microarrays and a quantitative polymerase chain reaction are sensitive, but not capable of identifying the post-transcriptional modifications of miRNA. Here we report a capillary electrophoresis-mass spectrometry (CE-MS) method, which performs a multiplex, direct analysis of miRNAs from biological samples. Using the CE-MS method, we detected two endogenous human circulating miRNAs, a 23-nucleotide long 5'-phosporylated miRNA with 3'-uridylation (iso-miR-16-5p), and a 22-nucleotide long 5'-phosporylated miRNA (miR-21-5p) isolated from B-cell chronic lymphocytic leukemia serum. The CE separation and following MS analysis provides label-free quantitation and reveals modifications of miRNAs. MicroRNA profiling of serum samples with CE-MS has the potential to be a versatile and minimally invasive bioassay that could lead to better clinical diagnostics and disease treatment.

Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

“…An electrophoretic preconcentration CE-MS method for the analysis of miRNA, described elsewhere 21 , was adapted to our specific CE-MS setup. Separations were performed at 15°C in a 158 cm long (LT) × 50 µm i.d.…”

“…In this paper, for the first time, an SPE-CE-MS method for the purification, preconcentration, separation, and untargeted multiplex analysis of miRNAs from serum samples is described. The SPE-CE-MS analysis of miRNA was optimized using standards, and the figures of merit were compared with those of a previously developed sample stacking CE-MS method 21 . Subsequently, the applicability of the developed SPE-CE-MS method was demonstrated by the analysis of serum samples from healthy controls and patients with chronic lymphocytic leukemia (CLL), the most common type of leukemia in adults 37 .…”

Recent advances in CE and microchip‐CE in clinical applications: 2014 to mid‐2017

“…Zeng et al. performed metabolomics analysis on a total of 183 human serum samples derived from patients with hepatocellular carcinoma by CE‐TOF‐MS. The authors claim that the technique was able diagnose cancer patients from noncancer controls as well as cancer patients from those with precancerous cirrhosis.…”

Future directions of extracellular vesicle-associated miRNAs in metastasis

“…The fact that miRNAs are found very close to these organelles gives a possibility that post-transcriptional miRNA modifications may be happening in this place. MicroRNAs are modified through a series of processing events after transcription like 5'-end phosphorylation, 3'-end adenylation or uridylation, and terminal nucleotide deletion (49). The study of Salzman et al showed that miR-34, a tumorsuppressor miRNA that is important in cell survival and that is transcriptionally upregulated by p53 in response to DNA damage is found in a pool of mature miRNA in cells that lack a 5'-phosphate and is inactive.…”

“…In a different study, it was shown that posttranscriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for regulation of miRNA activity. For example, such modification in plants and C. elegans influence miRNA stability (49,50). In humans, miR-122 was shown to be adenylated by the RNA nucleotidyl transferase GLD-2, which resulted in an increase in the stability of the miRNA (51).…”