MicroRNA profiling in ischemic injury of the gracilis muscle in rats.
BACKGROUND
To profile the expression of microRNAs (miRNAs) and their potential target genes in the gracilis muscles following ischemic injury in rats by monitoring miRNA and mRNA expression on a genome-wide basis.


METHODS
Following 4 h of ischemia and subsequent reperfusion for 4 h of the gracilis muscles, the specimens were analyzed with an Agilent rat miRNA array to detect the expressed miRNAs in the experimental muscles compared to those from the sham-operated controls. Their expressions were subsequently quantified by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) to determine their expression pattern after different durations of ischemia and reperfusion. In addition, the expression of the mRNA in the muscle specimens after 4 h of ischemia and reperfusion for 1, 3, 7, and 14 d were detected with the Agilent Whole Rat Genome 4 x 44 k oligo microarray. A combined approach using a computational prediction algorithm that included miRanda, PicTar, TargetScanS, MirTarget2, RNAhybrid, and the whole genome microarray experiment was performed by monitoring the mRNA:miRNA association to identify potential target genes.


RESULTS
Three miRNAs (miR-21, miR-200c, and miR-205) of 350 tested rat miRNAs were found to have an increased expression in the miRNA array. Real-time RT-PCR demonstrated that, with 2-fold increase after 4 h of ischemia, a maximum 24-fold increase at 7 d, and a 7.5-fold increase at 14 d after reperfusion, only the miR-21, but not the miR-200c or miR-205 was upregulated throughout the experimental time. In monitoring the target genes of miR-21 in the expression array at 1, 3, 7, 14 d after reperfusion, with persistent expression throughout the experiment, we detected the same 4 persistently downregulated target genes (Nqo1, Pdpn, CXCL3, and Rad23b) with the prediction algorithms miRanda and RNAhybrid, but no target gene was revealed with PicTar, TargetScanS, and MirTarget2.


CONCLUSIONS
This study revealed 3 upregulated miRNAs in the gracilis muscle following ischemic injury and identified 4 potential target genes of miR-21 by examining miRNAs and mRNAs expression patterns in a time-course fashion using a combined approach with prediction algorithms and a whole genome expression array experiment.
Hsieh
Ching-Hua
CH
Department of Plastic and Reconstructive Surgery, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Taiwan. m93chinghua@gmail.com
Jeng
Jonathan Chris
JC
Jeng
Seng-Feng
SF
Wu
Chia-Jung
CJ
Lu
Tsu-Hsiang
TH
Liliang
Po-Chou
PC
Rau
Cheng-Shyuan
CS
Chen
Yi-Chun
YC
Lin
Chia-Jung
CJ
D000818:Animals; D004195:Disease Models, Animal; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation / Q000235:genetics*; D055106:Genome-Wide Association Study; D007511:Ischemia / Q000235:genetics* / Q000378:metabolism* / Q000601:surgery; D035002:Lower Extremity / Q000098:blood supply / Q000601:surgery; D008297:Male; D035683:MicroRNAs / Q000235:genetics*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism; D018482:Muscle, Skeletal / Q000098:blood supply* / Q000378:metabolism* / Q000601:surgery; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation / Q000235:genetics
D035683:MicroRNAs; D009124:Muscle Proteins