Abstract
MicroRNA (miRNA/miR) 5'‑isoforms (5'‑isomiRs) differ from canonical sequences registered in the microRNA database in the length of their 5' ends. The 'seed sequence' of miRNAs that bind to target mRNAs is 2‑8 nucleotides from the 5' end; thus, shifts at the 5' end can cause a 'seed shift'. Accumulating data from miRNA deep sequencing have revealed that, in a substantial number of miRNAs, sequences corresponding to specific isomiRs, not the canonical form, are the most abundant. Studies have so far focused on circulating miRNAs as either markers or intercellular communication factors. miR‑1246 is abundant in the serum and is a candidate diagnostic and prognostic marker for esophageal squamous cell carcinoma, pancreatic cancer, hepatocellular carcinoma, colorectal adenocarcinoma and non‑small cell lung cancer (NSCLC). The present study analyzed the 5'‑end of serum miR‑1246 by fragment analysis and found that a 5'‑isomiR, which is two bases shorter than the canonical sequence, was the most abundant sequence in patients with NSCLC as well as healthy donors. To quantify the 5'‑isomiR, 5'‑isomiR‑specific primers based on primers for allele specific‑PCR were used, primarily because commercially available methods for miRNA Reverse transcription‑quantitative PCR cannot discriminate among sequences, especially those located at the 5' end of miRNA. The total miR‑1246 levels were significantly increased in patients with NSCLC; by contrast, the level of the canonical sequence was significantly decreased. Significant positive correlations were observed between the total miR‑1246 levels and the 5'‑isomiR levels, but not that of the canonical sequence. These results imply that the increase in levels of serum miR‑1246 in patients with NSCLC depends on increase of the 5'‑isomiR.