MicroRNA-206 Is Involved In Survival Of Hypoxia Preconditioned Mesenchymal Stem Cells Through Targeting Pim-1 Kinase

Background:Overexpression of Pim-1 in stem/progenitor cells stimulated cell cycling and enhanced cardiac regeneration in vivo. In addition, it was also found that the increased expression of Pim-1 could promote cardiomyocytessurvival and improve ventricular remodeling after acute myocardium infarction.We proposed that hypoxic preconditioning could increase survival of bone marrow mesenchymal stem cells(BMSCs or MSCs) via upregulation of Pim-1 and aimed to find out the microRNAs that modulate the expression of Pim-1.Methods :BMSCs were divided into four groups and respectively hypoxic preconditioned for 0h, 6h, 12 h,24h. The expression of pim-1 was detected by RT-qPCR and Western blot. Then, the best hypoxic preconditioning time was determined as 12 h and used as hypoxic treatment conditions in the following experiment, in which the expression of pim-1 is the most. Through a database query, miR-206 is predicted as one of the potential miRNAs that target Pim-1. RT-qPCR was performed to detect the expression of miR-206 in hypoxic MSCs and confirm the change of Pim-1 expression in MSCs after be transfected with miR-206 mimics and inhibitors.. Luciferase activity assay further was next used to further confirm the target relationship between mi R-206 and Pim-1. In addition, gain and loss-of-function studies including Cell apoptosis, migration and mitochondrial membrane potential were conducted to evaluate the function changes of BMSCs with different treatment(MSCs、HP-MSCs、HP-MSCsmiR-206、HP-MSCsanti-miR-206、HP-MSCs anti-miR-206 + Pim-1 inhibitor). Female young SD rats weighing 200-250 g were were subjected to the surgery of LAD ligation to establish myocardial infarction model. Sex-mismatched transplantation of male donor cells was performed during the acute phase after permanent LAD coronary artery ligation. The animals were euthanized on day 4 after their respective treatment. The heart tissue samples were collected and sry-gene expression was detected to evaluate the the survival rate of BMSCs.Results: MSCs were subjected to hypoxia exposure. The expression of Pim-1 in MSCs was enhanced in a time-dependent manner, detected by qPCR and western blot. miR-206 is predicted as one of the potential miRNAs that target Pim-1. The expression of miR-206 was decreased in hypoxic MSCs and reversely correlated with Pim-1 expression. Luciferase activity assay further confirmed Pim-1 as a putative target of mi R-206. In addition, gain and loss-of-function studies with miR-206 mimics and inhibitors showed that inhibition of miR-206 in hypoxic MSCs promoted the migration ability of the cells, prevented cell apoptosis and protected membrane potential of mitochondria, while the benefits were all blocked by Pim-1 inhibitor. In an acute model of myocardial infarction, transplanted hypoxic MSCsshowed a significantly improved survival as compared with hypoxic MSCs overexpressing miR-206.Conclusions:Hypoxic preconditioning could increase survival of bone marrow mesenchymal stem cells via upregulation of Pim-1, and mi R-206 was one of the critical regulators in this process.