Precursor miR-886, a novel noncoding RNA repressed in cancer, associates with PKR and modulates its activity

Reduced expression of pre-miR-886 in cancer cell lines and patient specimens. (A) The stem–loop structures of pre-miR-886 (adapted from the miRbase: http://www.mirbase.org/). The mature miR-886-5p and -886-3p portions (top and bottom, respectively), registered in the miRbase from high-throughput sequencing data, are shaded in gray. (B) From the miRNA array data, miRNAs were sorted according to the expression ratio of H1299 to CRL2741, and the 12 miRNAs with the greatest decreases were tabulated as shown. miR-886-5p and -886-3p are highlighted in bold letters. (C) BLAT analysis (using the UCSC genome browser) of pre-miR-886 (with the mature miRNA portions gray highlighted) showing the alignment with genomic loci having identical or highly similar sequences. Mismatched nucleotides are highlighted in gray. The nucleotide coordinates are based on the February 2009 assembly (GRCh37/hg19). Northern probes and qRT-PCR primers are indicated by arrows. (D) Northern hybridization showing pre-miR-886 in indicated cell lines, using “miR-886-3p as” (panel C) as a probe. tRNA–Ala was measured as a loading control. “M” denotes 5′-³²P-labeled Decade markers (Applied Biosystems/Ambion); their molecular sizes in nucleotides are shown on the right. (E) qRT-PCR measurement of pre-miR-886 in indicated cell lines (x-axis), using “miR-886-5p sense” and “miR-886-3p as” as primers (panel C). Each value was normalized to that of snU6 (y-axis). For the five oral cell lines, arranged in order of increasing capability of tumorigenesis and metastasis, the value of OKT-TERT1 was set as 1. To validate qRT-PCR assays, H1299 and CRL2741 were included for comparison to panel D. In this pair, the value of CRL2741 was set as 1. (F) Northern hybridization of pre-miR-886 in three pairs of a prostate tumor specimen (T) and the surrounding normal tissue control (N), with 5S rRNA as a loading control. The blot was exposed to the phosphorimager Storm 860 (Molecular Dynamics) and each band was quantified with ImageQuant 5.0 software. The intensity of pre-miR-886 was normalized to that of 5S rRNA (y-axis).