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Fig. 1 | Clinical Epigenetics

Fig. 1

From: Splicing-dependent expression of microRNAs of mirtron origin in human digestive and excretory system cancer cells

Fig. 1

Identification of splicing-dependent miRNAs processed from mirtrons. a Schematic representation of exon–intron structures of analyzed human minigenes. Boxes and lines indicate exons and introns, respectively. Introns containing validated mirtronic miRNA hsa-mir-1226-3p or predicted miRNAs hsa-mir-1227-3p, hsa-mir-1229-3p, hsa-mir-1236-3p, and hsa-mir-1238-3p processed from conventional; hsa-miR-3064-5p and hsa-miR-6515-5p from 5′-tailed; and hsa-miR-3940-5p and hsa-miR-6850-5p from 3′-tailed mirtrons are depicted as red lines. WT, wild-type sequences, and MUT, minigenes carrying the double mutant with GT to CT and AG to AC changes (affected nucleotides are marked in red), in 5′-donor and 3′-acceptor splice sites, respectively. b Splicing analysis of minigene transcripts in transfected HCT116 cells. The position of oligonucleotides used for RT-PCR analysis of mRNA transcripts produced by minigene constructs is indicated by white arrows in section a. Control reactions, RT, were performed without prior reverse transcription to exclude DNA contamination. Unspliced form indicates transcript retaining mirtronic intron; spliced, intron is excised. TBP — loading control. c miRNA expression in colorectal cancer HCT116 cell line transfected with native (WT) or splicing-deficient (MUT) minigenes was analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Expression level of RNU48 was used as endogenous reference for data normalization. The experiments were performed in at least three biological replicates. The error bars represent calculated values for standard deviation

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