Identification Of MicroRNAs Involved In Mesenchymal Stem Cell Apoptosis

Bone marrow mesenchymal stem cells (MSCs) appear to be an appealing source for cardiac engraftment in the treatment of myocardial infarction (MI) because of the ease of harvest and expansion ex vivo. However, the low cellular survival rate after transplantation into an infracted heart within the first few days engenders only marginal functional improvement. Thus, it is necessary to reinforce MSCs against the arduous microenvironment incurred from ischemia, inflammatory response, and proapoptotic factors in order to improve the efficacy of cell therapy. Recent research has identified pivotal roles for microRNAs (miRNAs) in a large number of cellular processes as a post-transcriptional gene expression regulator, including apoptosis. Nevertheless, the most studies have focused on cancer cells as a model to investigate the mechanistic participation of apoptosis regulated miRNAs in their endeavor to escape death in the ischemic microenvironment. Moreover, little is known about the miRNA global change and function during MSC apoptosis.We performed several experiments to investigate the function of miRNAs during MSC apoptosis in vivo and in vitro. Our main work includes three aspects as follows:Part I Identification of microRNAs Involved in Hypoxia and Serum Deprivation (hypoxia/SD) Induced Apoptosis in MSCs1) To explore the function of miRNAs in MSC apoptosis, the preferentially expressed miRNAs in normal culture and in 6 h hypoxia/SD-treated MSCs were measured using the high throughput TLDA platform. It revealed that 60 miRNAs were differentially expressed (>1.9-fold change) in MSCs exposed to hypoxia/SD for 6 h. Among these 60,57 were up-regulated while only 3 were down-regulated.10 miRNAs were selected from the result of miRNA TLDA experiments for verification by single-well TaqMan qRT-PCR. As a result, we found that miR-21, miR-23a, miR-34a, miR-210 and miR-503 were all increased more than 1.5 times after exposure of MSCs to hypoxia/SD for 6 h and altered dynamically with different duration in hypoxia/SD.2) To investigate the potential functions of miRNAs in the apoptosis of MSCs, we performed over-expression and knockdown experiments by direct transfection of miRNA mimics or miRNA inhibitors. MSCs were exposed to hypoxia/SD for 6 h after transfection with miRNA mimics/inhibitors. Apoptosis of MSCs was measured by using FACS analysis after staining with Annexin V and PI. We found that down-regulation of miR-21, miR-23a, or miR-503 precipitated the apoptosis of MSCs. Over-expression of miR-21, miR-23a or miR-210, in contrast, promoted the survival of MSCs when these cells were exposed to hypoxia/SD.3) MSCs were treated with hypoxia/SD 6 h after over-expression of miR-21, miR-23a, miR-34a, miR-210 and miR-503, and then Caspase-3/7 activity was measured by using Caspase-Glo 3/7 reagent. Caspase-3/7 activity could be inhibited be miR-21, miR-23a and miR-210.4) We assessed mitochondrial membrane potential (??m) of MSCs after over-expression of miR-21, miR-23a and miR-210. In contrast to MSCs transfected with the mimics scramble group, cells transfected with the mimics of miR-21 and miR-23a partly prevented the fall in??m under hypoxia/SD.Part I investigation demonstrated that miRNA profiles were altered under hypoxia/SD treatment. Up-regulation of miR-21, miR-23a and miR-210 induced by hypoxia/SD may be involved in protecting MSCs against apoptosis by suppressing Caspase-3/7 activity. Among them, miR-21 and miR-23a augmented MSC survival partly by preventing the fall in??m under hypoxia/SD; however, miR-210 may resort to other pathways.Part II miR-21 Suppressed the Apoptosis Induced by Hydrogen Peroxide (H2O2) in MSCs1) Oxidative stress on the cell death of MSCs was mimicked in vitro by treating with H2O2 (200?M,4 h). The expression level of miR-21, miR-23a and miR-210 was detected by using TaqMan qRT-PCR, and miR-21 was up-regulated significantly under the treatment of H2O2.2) MSCs were exposed to H2O2(200?M,4 h) after transfection with miR-21, miR-23a and miR-210 mimics. Apoptosis of MSCs was measured by using FACS analysis after staining with Annexin V and PI. Lactate dehydrogenase (LDH) release level was measured by LDH assay kit. We found that apoptosis of MSCs could be suppressed by up-regulation of miR-21, not miR-23a and miR-210.3) Caspase-3/7 activity was measured by using Caspase-Glo 3/7 reagent after treated with H2O2 (200?M,4 h). We found that MSC apoptosis could be inhibited by over-expression of miR-21. It indicated that miR-21 might enhance MSC survival by suppressing Caspase-3/7 activity.4) PDCD4 is a gene target of miR-21, and we assessed the expression of PDCD4 in protein level by western blot. We found that H2O2 decreased PDCD4 expression, miR-21 inhibitor increased PDCD4 expression, and miR-21 mimics decreased PDCD4 expression. It suggested that PDCD4 related to miR-21-mediated anti-apoptotic effect on MSCs.Part II results shown that miR-21 was sensitive to H2O2 stimulation and participated in H2O2-mediated PDCD4 gene regulation and function modulation in MSCs. As an anti-apoptosis miRNA, miR-21 might be involved in H2O2 induced apoptosis as well as hypoxia/SD. It indicated that miR-21 might be an effective target to promote MSC survival after transplanting into myocardium.Part III miR-21 Epigenetic Modified MSCs Augmented Survival and Improved Heart Function1) Female SD rats were operated to induce coronary artery occlusion. Animals were randomly divided into four groups (n=6):(A) group:Sham; (B)-(D) groups, to receive intramyocardial injection with IMEM (30uL, MI/IMDM); male MSCs transfected with miRNA mimics (4x 106 per rat, MI/miRsc-MSCs) or male MSCs transfected with miR-21 mimics (4x106 per rat, MI/miR21-MSCs), respectively.2) The results demonstrated that miR-21 epigenetic modification improved graft MSC survival in ischemic myocardium assessed in a gender-mismatched transplantation model by real time-PCR, as well as by TUNEL assay.3) Effects of miR-21 modified MSCs on myocardial function in sham or MI hearts 4 weeks after therapeutic intervention. M-mode echocardiography measurements for left ventricular function demonstrated that LVEF, LVFS, LVEDd and LVESd were improved more in the MI/miR21-MSC group than in the MI/miRsc-MSC group at 4 weeks after MI. Compare with IMDM injected group (MI/IMDM group) both had significant improvements in cardiac function.Part III data suggested that miR-21 exerts both protective actions on MSC survival and enhancement on myocardial function. MiR-21 epigenetic modification may represent a novel and effective treatment strategy in cell transplantation.In summation, our present study explored the mechanism of MSC apoptosis involved in miRNAs. We also found miR-21 epigenetic modification could improve the survival and cardiac function. For the advantages of miRNAs, developing miRNA epigenetic MSCs has a wide application prospect in MSC transplant to treat MI.