In this study, we demonstrated that 3 miRNAs (miR-21, miR-200c, and miR-205) were significantly upregulated in a different pattern in the gracilis muscles following ischemic injury. Ischemia for only 1 h was sufficient to induce the expression of miR-21 and miR-200c during the reperfusion stage; and 2 h of ischemia were required to induce the expression of miR-205. Our data also showed that miR-200c and miR-205 were actively regulated after ischemia but their expression pattern changed after reperfusion, indicating their temporal expression during ischemic injury. In addition, with a yet to be determined mechanism, we noted a significant re-expression of miR-205 at 7 and 14 d after the injury. In contrast, the expression of miR-21 gradually increased to its maximum level at 7 d and persisted throughout the experiment for at least 14 d after the ischemic injury. This finding might imply an important role of miR-21 during ischemic injury in the muscle.
The overexpression of miR-21 has been shown to be in a number of medium-scale and high-scale profiling experiments that were designed for the detection of miRNAs that are dysregulated in cancer [
27]. In addition, miR-21 has been reported to have anti-apoptotic properties in cancer cells [
28,
29]. It has been reported that miR-21 protects against the hydrogen peroxide-induced injury of cardiac myocytes via its target gene, the repressor gene programmed cell death 4 (
PDCD4), and the AP-1 pathway [
30]. The hydrogen peroxide-induced cardiac cell death and apoptosis were increased by a miR-21 inhibitor and was decreased by pre-miR-21 transfection [
30]. In addition, a significant induction of miR-21 was noted in the heart following whole body heat-shock [
19]. The injection of chemically synthesized exogenous miR-21 significantly reduced infarct size in the heart which was blocked with a miR-21 inhibitor [
19]. In an investigation of rat hearts at 6 h after acute myocardial injury, miRNA signatures in the early phase revealed that, among multiple aberrantly expressed miRNAs, miR-21 was significantly downregulated in infarcted areas, but was upregulated in border areas [
31]. Remarkably, the downregulation of miR-21 in infarcted areas was inhibited by ischemic preconditioning, a known cardio-protective method. In addition, adenoviral overexpression of miR-21 had a protective effect on myocardial infarction by decreasing the infarct size by 29% at 24 h [
31]. In this study, we found 4 potential target genes (
Nqo1,
Pdpn,
CXCL3, and
Rad23b) of miR-21 during skeletal muscle ischemic injury. The database of experimentally validated miRNA target genes, MiRecords [
32], lists 26 validated target genes of miR-21 in humans (
TPM1,
CDK6,
TIMP3,
PDCD4,
SERPINB5,
NFIB,
CDKN1A,
FAS,
FAM3C,
HIPK3,
PRRG4,
ACTA2,
BTG2,
BMPR2,
SESN1,
IL6R,
SOCS5,
GLCCI1,
APAF1,
SLC16A10,
SGK3,
RP2,
CFL2,
RECK,
MTAP,
SOX5) and only 2 validated target genes in the rat (
ITGB1,
Tagln). Interestingly, the 4 genes that we identified in this study did not match with the already validated target genes in humans or rats. Among these 4 identified target genes, podoplanin (Pdpn) is a transmembrane glycoprotein and is expressed in many normal human tissues including the skeletal muscle [
33]. Pdpn is widely used as a specific marker for lymphatic endothelial cells and lymphangiogenesis in many species because it is expressed on lymphatic but not on blood vessel endothelium [
33]. Deficiency of Pdpn results in congenital lymphedema and impaired lymphatic vascular patterning [
34]. With the postfix L (for ligand) or postfix R (for receptor), CXCL and CXCR are used as ligands and receptors of the CXC chemokine family which exhibits both angiogenic and angiostatic properties [
35]. While chemokines can exert a pro-angiogenic effect via recruitment of inflammatory cells,
CXCL3 is induced in the human perihematomal tissue [
36] and can mediate angiogenesis in the absence of preceding inflammation [
35]. The nucleotide excision repair (NER) protein, Rad23b, was found to be downregulated in hypoxic cancer cells [
37]. Hypoxia can also promote genetic instability by affecting the DNA repair capacity, including NER which primarily focuses on helix-distorting injuries [
38]. The downregulation of Rad23b in hypoxic cancer cells could be partially reversed by antisense inhibition of miR-373 [
37], indicating a key role of miR-373 in modulating the basal expression of Rad23b. The effect of anti-miR-373 activity in normoxia is more profound than in hypoxia; thus, it has been suggested that there might be other mechanisms that regulate the expression of Rad23b [
37]. In reviewing the literature, no direct linkage between these 4 identified target genes and the muscle ischemic injury was found. Only the detoxification enzyme NAD(P)H: quinone oxidoreductase 1 (Nqo1) could be correlated to the ischemic injury in renal tissue [
39] and primary cultures of rat cortex [
40], but with different outcomes regarding the cytoprotective effect. As a detoxification enzyme Nqo1 catalyzes the 2-electron reduction of quinoid compounds to the readily excreted hydroquinones, thereby preventing the generation of reactive oxygen species and protecting cells against oxidative damage [
41]. The expression of the
Nqo1 gene is primarily regulated via antioxidant response element sequences in the promoter region. Reoxygenation-specific activation of the antioxidant transcription factor Nrf2 mediates the cytoprotective gene expression during ischemia-reperfusion injury [
42]. However, some authors suggested a deteriorating rather than a protective factor of Nqo1 in the progression of neuronal cell death, as inhibition of Nqo1 by various inhibitors protects against neuronal damage in vitro and following cerebral ischaemia in vivo [
40]. Because bioinformatic analysis has indicated that miRNAs frequently interact with transcription factors in feedback and feedforward loops to regulate their target genes [
43,
44]; therefore, further experiments are required to elucidate the function and role of these potential target genes and the upregulated miR-21, as well as miR-200c and miR-205, in ischemic injury.
The pneumatic tourniquet is frequently used in surgery to acquire a bloodless operative field. To prevent the ischemia-reperfusion injury, the maximal allowable ischemic time of the pneumatic tourniquet is 2 h [
45,
46] Although minor histological changes in muscle started to appear after about 35-40 minutes, there was no clinical evidence of irreversible muscle damage within 2 h [
45‐
47]; this is also the reason why most of the article studying the ischemic injury of skeletal muscle would choose the ischemic time at 4 h or a longer ischemic time. Whether there is a different miRNAs expression profile after a shorter time of ischemia such as 1 h or 2 h is unknown, but if these miRNAs are not up-regulated in a longer hour ischemia, the investigation of these targets may be devoid of clinical importance or attention. Therefore, in this study, we were not intended to profile the miRNAs expression with a miRNA array at a shorter time, like 2 h, of ischemia. However, as shown in the Figure
2, in the investigation of minimal ischemic time that would induce the expression of these 3 miRNAs, we had demonstrated that 1 h of ischemia was able to increase the expression of miR-21 and miR-200c and 2 h of ischemia would increase the expression of these three miRNAs, implying the epigenetic regulation could be induced by a shorter time of ischemia before a remarked pathophysiologic change could be observed by a longer time of ischemia.
Bioinformatic algorithms remain the principal means of predicting targets of specific miRNAs. These algorithms take into account numerous parameters that influence miRNA/target interactions, including seed match (complementarity), 3'-UTR seed match context, seed match conservation, favorability of free energy binding, AU content, and binding site accessibility [
48]. To identify miRNAs that regulate mRNAs, one needs to co-analyze the changes in miRNA and mRNA expressions. The investigation of the mRNA:miRNA association monitors the miRNA and mRNA expression on a genome-wide basis and provides an analytical approach to reveal the target genes of the miRNA [
49,
50]. However, some limitations still remain for the combined approach method that we described here. First, it might be overly simplistic to correlate miRNAs and their predicted targets primarily on the basis of the number of consensus sites in the 3'UTR because an exact match to the sequence of the seed region is not required. For example, miRanda typically produces more potential targets than other programs, but a large number of false targets would seriously limit the value of the output information [
51]. In contrast, some available programs with stricter criteria, e.g., the prediction algorithms PicTar, TargetScanS, and MirTarget2 have only partially overlapping predicted targets for the same miRNA and produce smaller data sets than miRanda [
52]. In this present study, in order to reduce the noise from the calculation of the correlation between miRNA and mRNAs (based on the time-course expression values), the inclusion of downregulated genes at all 4 time points might have been too strict to acquire potential target genes. In addition, it might have produced a smaller number of predicted targets after correlation with the stricter prediction algorithm considering the accuracy and reproducibility of the whole genome array. For example, no target genes were found for the combination with the prediction algorithms PicTar, TargetScanS, and MirTarget2. Due to the differences among databases and because there is no clear superior method, a further gain-of-function or loss-of function experiment would be helpful to elucidate the role of the identified target genes and that of each associated miRNA. Furthermore, it has been reported that more than a third of those translationally repressed target genes always displayed detectable mRNA destabilization [
14]. However, the extent of miRNA function in animal cells has largely been studied by mRNA microarray profiling assuming that miRNA function leads to reduced mRNA levels, which may not be always the case. There might be some unidentified target genes that are repressed only in the translation process but have not been subjected to mRNA degradation.