| The expression of members of miR-17-92cluster in wild type mice myocardium and the changes during ischemiaBackground-It is known that miR-17-92cluster is very important and vital for the survival of mice embryos and newborns. And it is verified that miR-17-92cluster is high expressed in canine. But the effect of miR-17-92cluster in myocardial ischemia and reperfusion is unknown. Thus, to investigate the expression of members of wild type mice myocardium and the changes during ischemia is urgent.Methods and Results-To harvest the hearts from C57BL/6wild type mice and extract total RNA. Using qRT-PCR to measure the expression of members of miR-17-92cluster. After permenant ligation of left anterior descending, the mice hearts were harvested and the miR expression were measured as previously1day or4days or7days post-operatively. The miR-92a is highest expressed in normal mice hearts. MiR-19a/19b family is lower, miR-17/20a is still lower and miR-18a is lowest. They constitute62%,21%,16%and1%of the entire cluster expression, respectively. After operation, the expression of all the members except miR-18a were reduced to30%-50%that pre-operatively(p<0.05). Although miR-18a was not down-regulated, its expression constituted only2%-3%of entire cluster expression post-operatively.Conclusions-the members of miR-17-92cluster express diversely in mice heart. MiR-92a, miR-19a/19b, miR-17/20a are high expressed while the miR-18a is low expressed. All the members are down-regulated except miR-18a1day to1week after operation. This suggests the miR-17-92may play a role in myocardial ischemia and reperfusion. PART2The role of down-regulated expression of miR-17-92cluster in myocaxdial ischemia and reperfusionBackground-It is verified in part1that miR-17-92cluster was down regulated during myocardial ischemic injury in mice. Therefore, to investigate the role of miR-17-92cluster in myocardial ischemia and reperfusion, we cultured condition-gene knock-out transgenic mice.Methods and Results-to crossbreed miR-17-92cluster floxed (miR-17-92F/F). mice and a MHC-MerCreMer (α MHC-MCM) mice to get double transgenic mice (a MHC-MCM/miR-17-92F/F). At the age of6weeks to8weeks, the mice were separated to the control and the study groups. The control group were fed corn oil while the study group were fed tamoxifen to knock out the miR-17-92cluster in cardiomyocytes. Then ischemia and reperfusion procedure was performed on both groups. After24hours, they were sacrificed to get TTC-Phthalocyanine Blue staining or western blot. The expression of members of miR-17-92cluster in study group were significantly reduced to30%-50%(p<0.05) that of control group. After reperfusion for24hours, the ratio of infarct area/risk area was significantly higher in study group (n=10) compared with the control group(n=7)(63.6±3.9%vs41.0±3.1%, p<0.05). Western blot revealed that the phosphorylation was significantly inhibited in raf-l-Mek1/2-MAPK1pathway and PI3K/Akt pathway. The heme oxygenase-1increased significantly in control group but not in the study group. In the growth factors withdrawal apoptosis pathway, the anti-apoptosis factor bcl-2was reduced and the pro-apoptosis factors bak, bax increased in the study group. And these led to cleaved PARP and caspase3increase. Conclusion-the down-regultion of miR-17-92cluster increases the injury from myocardial ischemia and reperfusion. The function is achieved through inhibition of phosphorylation of Raf-1/Mek1/2/Erk1/2pathway and PI3K/Akt pathway, through inhibition of synthesis of heme oxygenase-1and through the increase of apoptoisis induced by reduction of anti-apoptosis factors and increase of pro-apoptosis factors. The role of down-regulated expression of miR-17-92cluster in cardiac progenitor cells during hypoxia and reperfusionBackground-We have got in vivo results from part2. However, the expression of members of the miR-17-92cluster just down-regulated50%-70%. To get more efficient miR-17-92cluster knocked-out cells, we have to use miR-17-92F/F cardiac progenitor cells (CPCs) and adenovirus ad-cre, since the cardiomyocytes can just survive about3days in vitro and do not have adequate time to eliminate the toxicity of cre recombinase.Methods and Results-to select the c-kit positive cells as miR-17-92F/F CPCs with magnetic beads from miR-17-92F/Ftransgenic mice heart derived cells. The CPCs were divided into to two groups, one transfected with adenovirus ad-GFP as control, the other transfected with adenovirus ad-cre as study group. After2weeks, miRNA was measured to identify the gene knock-out results. Later, the two groups were treated with hydrogen peroxide and measured the caspase3/7. Other cells were treated with hypoxia-reperfusion and measured the changes in protein with western blot. After ad-cre expression, we found the expression in study group reduced significantly, miR-17/20a0.0944±0.004, miR-18a0.240±0.097, miR-19a/b0.234±0.019, miR-92a0.023±0.007(p<0.05). The caspase3/7were1.035±0.0187VS1.253±0.077(p<0.05) for the control and study groups treated with600uM H2O2and1.242±0.016VS1.521±0.037(p<0.05) in those treated with1100uM H2O2. These results suggested the study group had more apoptosis after injury. The western blot confirmed the reduced phosphorylation in the members of raf-1-Mek1/2-MAPK1pathway and the PI3K/Akt pathway. The hypoxia-inducible factor1a increased in the study group while this did not lead to high expression of Heme Oxygenase-1. On the contrary, the Heme Oxygenase-1was lower in the study group. For the growth factors withdrawal apoptosis pathway, bcl-2decreased and Mcl-1increased as anti-apoptosis factors and the pro-apoptosis factors such as bim, bad and bak increased. And all these finally led to increased cleaved caspase3and caspase9.Conclusion-the down-regultion of miR-17-92cluster increases the injury from hypoxia and reperfusion in CPCs. The function is achieved through inhibition of phosphorylation of Raf-1/Mekl/2/Erkl/2pathway and PI3K/Akt pathway, through inhibition of synthesis of heme oxygenase-1and through the increase of apoptoisis induced by both reduction and increase of anti-apoptosis factors and increase of pro-apoptosis factors. The novelty of this study1. This is the first report that miR-17-92plays a role in myocardial ischemia and reperfusion. And it was proved in vivo that miR-17-92knocked down can increase the injury from ischemia and reperfusion.2. This is the first report that the miR-17-92knocked down can inhibit the phosphorylation of raf-1-Mek1/2-MAPK1pathway.3. It is known that HIFla3’UTR has target of miR-17, so the HIF1a expressed higher in study group. However, the down stream Heme oxygenase decreased. This told us the effect of microRNAs can not be determined by the target. It can be the opposite under some conditions.Deficiency and modificationDeficiency-no animal model of miR-17-92cluster over-expression.Modification-culture of transgenic mice of condition over-expression of miR-17-92cluster. |
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