Bone Marrow Mesenchymal Stem Cell Modified With Mir-499Activate Wnt/β-catenin Pathway And Enhance Cardiac Function In Rat Myocardial Infarction Model

BackgroundIn recent years, with the improvement of living standards and lifestylechanges, the incidence of myocardial infarction is increasing year by year in China,myocardial infarction is one of the most common cause of death and disability ofresidents. Although considerable progress has maken in the diagnosis andtreatment to myocardial infarction, implement of percutaneous coronaryintervention (percutaneous coronary intervention, PCI) and new drugs significantlydeclined the mortality of myocardial infarction, however, these treatment measuresdo not resolve the fundamental issue, which is absolute decline in the number ofcardiomyocytes caused by myocardial infarction. Bone marrow mesenchymal stemcells (bone marrow mesenchymal stem cell, BM-MSC) transplantation is anexciting orientation in treatment of myocardial infarction. Simple BM-MSCtransplantation has no significantly long-term benefit in treatment of myocardialinfarction, and the endpoint of all-cause death is not changed.In order to clarify the mechanism in miR-499promote BM-MSCdifferentiation into cardiomyocytes, and over-expression of miR-499in BM-MSCstransplantat into myocardial infarction border area whether enhance cardiacfunction, decline myocardial infarct size and improve BM-MSC survival in ratmyocardial infarction model. We conducted the following experiments. Method1. Isolation,culture,purification and identification of rat bone marrowmesenchymal stem cells;2. Use lipofecatemine-2000to transfect rno-miR-499mimics and inhibitorinto bone marrow mesenchymal stem cells to verify the target gene ofmiR-499;3. Use lentivirus-vectors(carries with GFP) to overexpress miR-499to verifybone marrow mesenchymal stem cells whether differentiate intocardiomyocyte-like cells and its possible mechanisms;4. Real-time quantatitive PCR to verify the expression of miR-499targetgenes and cardiac-specific gene NXK2.5in BM-MSC;5. Western blot to detect volume of cTnI and degree of β-cateninphosphorylation in BM-MSC after lentivirus transfected;6. Produce rat model of myocardial infarction, and then transplant of bonemarrow mesenchymal stem cells;7. Use Echocardiography to detect cardiac function of animal model;8. Use Hemodynamic to detect left ventricular function of animal model;9. Rats were sacrificed and heart slices, infarct size measurements;10. Use Real-time quantative PCR to detect transplanted bone marrowmesenchymal stem cells survived amount in border area of myocardialinfarction.Results1. The whole bone marrow culture method can obtain bone marrowmesenchymal stem cells, inverted phase contrast microscope fusiformdistribution of spiral polarity,and CD29(+), CD44(+), CD90(+), CD31(-), CD45(-) were confirmeded by flow cytometry.2. Use Lipofacatemine-2000transfected with miR-499mimic can decreaseexpression of sox6in BM-MSC; whlie transfected with miR-499inhibitor,can increase expression of sox6;3. Five days after BM-MSC overexpression of miR-499, NKX2.5increased2.23folds compared with the control group;4. MiR-499reduced the degree of β-catenin phosphorylation and activeWnt/β-catenin signal pathway, xav939play an inhibitor role in this process;5. Bone marrow mesenchymal stem cell overexpressed of miR-499enhanceLVEF,LVFS in Rat model of myocardial infarction;6. Bone marrow mesenchymal stem cell overexpressed of miR-499canreduce myocardial infarct size in rat model;7. Bone marrow mesenchymal stem cells overexpressed of miR-499transplantation can improve±dP/dt in rat myocardial infarction model;8. Overexpressed of miR-499has no significant benefit in BM-MSCsurvival;Conclusion1. Overexpression of miR-499can promote bone marrow mesenchymal stemcells differentiate into cardiomyocyte-like cells;2. MiR-499target gene is sox6;3. miR-499plays role in differetiation by activate the Wnt/β-cateninsignaling pathway,XAV939acts as an inhibitor in the process of BM-MSCdifferentiation into cardiomyocyte induced by miR-499;4. Transplantion with bone marrow mesenchymal stem cell overexpressedwith miR-499can enhance cardiac function, and also reduce myocardialinfarct size in rat myocardial infarction model.