Inhibition Of MiR-155-5p Rejuvenates Aged Mesenchymal Stem Cells And Enhances Cardioprotection Following Myocardial Infarction

Objective: Myocardial infarction(MI)is still the main cause of morbidity and mortality worldwide,particularly in elderly patients.Over the past decades,mesenchymal stem cell(MSC)-based therapy has been intensively investigated and shown promising results in the treatment of MI.Nonetheless,accumulating data have demonstrated that discrepancy in the effects of MSCbased therapy in the treatment of MI may due to senescence-induced alterations in their function.Micro RNAs(mi RNAs)play crucial roles in regulating senescence of MSCs,however,the underlying mechanisms remain unclear.This study aimed to detect the senescent phenotype of AMSCs,to figure out if the mechanism of AMSCs senescence can be put down to imbalance of mitochondrial dynamics,to figure out if mi R-155-5p induces increased mitochondrial fusion and decreased mitochondrial fission and finally causes AMSCs senescence,to investigate if mi R-155-5p regulates MSCs senescence via Calcium-binding protein 39(Cab39)/ Adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK)signaling pathway and whether inhibition of mi R-155-5p can rejuvenate aged-MSCs(AMSCs)to enhance the therapeutic efficacy for MI.Methods:Firstly,young-MSCs(YMSCs)and AMSCs were isolated from young and aged donors,respectively.The surface markers of MSCs were evaluated by flow cytometry.To figure out whether MSCs were senescent with age,the differentiation capacity of MSCs into adipocytes and osteocytes were measured.Proliferation,migration ability and senescence degree of MSCs were detected by cell counting kit 8(CCK8)assay,Ki67 proliferation assay,Transwell assay,senescence-associated ?-galactosidase(SA-?-gal staining)and the expression level of senescenceassociated protein p53 and p21.Then,the morphology of mitochondria was examined by Mitotracker staining.Mitochondrial structure with details was observed using electron microscope.Western blotting showed the expression level of p-Drp1 ser616,Mfn1 and Mfn2.To investigate whether mi R-155-5p regulates MSC senescence and explore the mechanisms inside,we carried out real-time quantitative PCR(q RT-PCR)to measure mi R-155-5p expression of serums from the young and aged,as well as YMSCs and AMSCs.YMSCs were transfected with mi R-155-5p mimic and AMSCs were transfected with mi R-155-5p inhibitor,respectively.The level of mi R-155-5p was determined by q RT-PCR.The cellular senescence of MSCs was evaluated by SA-?-gal staining.Next,we treated YMSCs with mi R-155-5p mimic and Mfn2-si RNA to figure out if mi R-155-5p induce MSCs senescence via activating mitochondrial fusion.Furthermore,we treated mi R-155-5p inhibitor and Drp1 inhibitor,Mdivi 1 to figure out if inhibition of mi R-155-5p to alleviate the senescence of AMSCs via increased expression of pDrp1.We aimed to determine whether mi R-155-5p regulates mitochondrial dynamics via AMPK signaling pathway.We used Target Scan to predict the target genes of mi R-155-5p and found that a potential binding sequence at the 3'UTR of Cab39.Dual-luciferase reporter gene assay further comfirmed the relationship of mi R-155-5p and Cab39.Then,AICAR,an AMPK activator treatment with mi R-155-5p mimic to figure out if the activation of AMPK signaling pathway will partially reverse the downregulation of p-AMPK,p-Drp1 and upregulation of Mfn2.To examine whether inhibition of mi R-155-5p in AMSCs can improve the therapeutic effects of MSCs,we transplanted YMSCs,AMSCs and anti-mi R-155-5p-AMSCs into the peri-infarct region of aged mice model of MI.The heart function and infraction area of mouse from different groups were evaluated by echocardiography and Masson's trichrome staining,respectively.Next,we examined the survival of MSCs at 28 days post transplantation by HNA staining.To evaluate the anti-apoptotic effects of MSCs transplantation,the apoptosis of cardiomyocytes in the ischemic area was determined by TUNEL staining and to determine the angiogenic effects of MSCs transplantation,the arteriole and capillary densities were examined by ?-SMA staining and CD31 staining in the mouse heart at 28 days post transplantation,respectively.Results: Compared with YMSCs,AMSCs exhibited increased cellular senescence as evidenced by elevation of SA-?-gal activity,decreased differentiation ability,decreased proliferative capacity,decreased migration ability and decreased secretion of angiogenic factors.Next,imbalance of mitochondrial dynamics is existed in AMSCs.The expression level of mi R-155-5p was much higher in both serum and MSCs from aged donors than young donors.Upregulation of mi R-155-5p in YMSCs led to increased cellular senescence whereas downregulation of mi R-155-5p decreased AMSCs senescence.Mechanistically,mi R-155-5p,via AMPK signaling pathway,inhibited mitochondrial fission and increased mitochondrial fusion in MSCs,thereby resulting into cellular senescence by repressing the expression of Cab39.These effects were partially reversed by AMPK activator or mitofusin2-si RNA(Mfn2-si RNA).As a result,transplantation of anti-mi R-155-5p-AMSCs led to improved cardiac function in aged mice model of MI compared with AMSCs by enhancing angiogenesis and promoting cell survival.Conclusion: In summary,our study shows that AMSCs exhibits increased cellular senescence;mitochondrial fusion contributes to senescence of AMSCs;mi R-155-5p mediates MSCs senescence via regulating Cab39/AMPK signaling pathway and that mi R-155-5p is a novel target to rejuvenate AMSCs and enhance the cardiopretective effects by enhancing angiogenesis and promoting cell survival.