Regulatory Role Of MicroRNA-34a In The Senescence Of Rat Bone Marrow Mesenchymal Stem Cells By Targeting Nampt

The organism aging,as a complex biological process,has been actually attributed to the stem cell senescence.Mesenchymal stem cells(MSCs)are a kind of adult stem cells with unique advantages,thereby they have become promising seeding cells,which are indispensable for stem cell-based therapeutic strategies and the maintenance of tissue homeostasis and regeneration.However,MSCs will inevitably undergo senescence whether in the natural aging process of individuals or in the process of cultivation in vitro.Therefore,how to postpone or reverse the MSC senescence is one of the essential scientific issues needed further study and exploration urgently.In the previous study,we found that nicotinamide phosphoribosyltransferase(Nampt)could affect the senescence of MSCs by mediating intracellular NAD~+synthesis and further regulating Sirt1 activity in the model of MSC replicative senescence in vitro.So we termed this regulatory process as Nampt-NAD~+-Sirt1 axis.Since Nampt is an important regulatory factor implicated in delaying MSC senescence,we wonder what factors could be involved in the regulation of Nampt and thus affect MSC senescence.The senescence of MSCs is not only modulated by genetics but also controlled by epigenetics.As one of the vital mechanisms of epigenetic regulation,microRNAs(miRNAs)participate in regulating the various biological processes,such as cell proliferation and differentiation,cellular senescence,energy metabolism and so on.Mounting studies have shown that microRNA-34a(miR-34a)is one of the important members of senescence-associated microRNAs(SA-miRNAs).The bioinformatics prediction demonstrated that miR-34a has potential binding sites with the 3'-UTR region of Nampt,thereby we further speculated that mi R-34a is able to regulate MSC senescence by targeting Nampt.In this study,both the natural aging rat model in vivo and MSC senescence model in vitro including MSC natural aging and MSC replicative senescence were used.RT-qPCR was applied to detect the expression of miR-34a in different aging models to figure out the alteration of miR-34a expression levels.Then the regulatory roles of miR-34a in MSC senescence were investigated by altering miR-34a expression via lentiviral transduction.Furthermore,we determined miR-34a regulation on MSC senescence from the morphological,functional and molecular characteristics respectively.What's more,the bioinformatics analysis and dual-luciferase reporter gene assay were utilized to validate that Nampt is a direct target of miR-34a.Then Nampt expression at both mRNA and protein levels were detected,and miR-34a rescue assay was performed to further verify miR-34a regulation on MSC senescence via targeting Nampt.Eventually the regulatory mechanisms by miR-34a-Nampt was explored by examining intracellular NAD~+concentration and Sirt1 activity.The results are as follows:1.Compared to young rats(1-2 months old,Young,Y),aged rats(15-18 months,Old,O)showed marked signs of aging.Senescence-associated?-galactosidase(SA-?-gal)staining in kidney and liver tissues from the aged rats were clearly shown to be blue-stained.Thus,the natural aging rat model in vivo was established.MSCs at passage 3(P3MSCs,OMSCs)were obtained from young and aged rats in natural aging rat model by whole bone marrow adherent culture method.P10MSCs were obtained from P3MSCs by the serial cultivation in vitro.MSC senescence model in vitro was thereby established.Compared with P3MSCs(young cells),both P10MSCs(replicative senescent cells)and OMSCs(natural senescent cells)exhibited senescent morphological features with enlarged cell areas,reduced cell aspect ratio and enhanced SA-?-gal activity.2.In the natural aging rat model,when compared to the young rats,miR-34a expression was obviously increased in all the tissues from the aged rats,especially in the heart,brain and kidney.And in the MSC senescence model,miR-34a expression showed a significant age-dependent and passage-dependent elevation.3.Lentiviral-mediated miR-34a overexpression made young P3MSCs present senescence-like alterations with enlarged cell areas and decreased cell aspect ratio.The cell growth became slower and cell proliferation activity was decreased,whereas the population doubling time was significantly prolonged.The majority of cells were arrested at G1 phase.And osteogenic differentiation was declined.In addition,miR-34a over-expression increased SA-?-gal activity and up-regulated the expression levels of senescence-related factors.Accordingly,miR-34a overexpression promotes senescence of MSCs.4.Inhibition of miR-34a expression by lentivirus transduction led to the morphological alterations in the senescent cells including P10MSCs and OMSCs from senescence-like enlarged cells to slender spindles with decreased cell areas and increased cell aspect ratio.The cell growth became faster and cell proliferation capability was enhanced,whereas the cell population doubling time was shortened markedly.The percentage of cells arrested at G1 phase was reduced and osteogenic differentiation was improved.What's more,SA-?-gal activity was dramatically declined and the expression levels of senescence-related factors were obviously down-regulated.miR-34a silcence thus suppresses the occurrence of MSC senescence.5.Bioinformatics analysis revealed that there are potential complementary binding sites between miR-34a and the 3?-UTR region of Nampt mRNA.Dual-luciferase reporter gene assays futher confirmed that miR-34a can directly target Nampt.6.miR-34a overexpression could down-regulate Nampt expression in P3MSCs at both mRNA and protein levels,while miR-34a suppression could up-regulate Nampt expression in both P10MSCs and OMSCs.Co-overexpression of miR-34a and Nampt in P3MSCs could not only rescue the inhibiton effects of miR-34a on Nampt,but also attenuate the enhancement of SA-?-gal activity caused by miR-34a overexpression.The above results further confirmed that miR-34a regulated MSC senescence via directly targeting Nampt.7.miR-34a overexpression markedly contributed to the reduction of the intracellular NAD~+amount and Sirt1 activity in P3MSCs,while miR-34a inhibition significantly increased the intracellular NAD~+content and Sirt1 activity in both P10MSCs and OMSCs.In summary,miR-34a plays the regulatory roles in MSC senescence,which may be achieved by directly targeting Nampt and then influencing NAD~+-Sirt1 axis.This study reveals the molecular mechanisms of miR-34a-Nampt network modulating MSC senescence from the perspective of epigenetics.It will not only provide the theoretical basis and experimental data for the study of molecular mechanisms of stem cell senescence and the new targets for activating senescent stem cells to rejuvenate,but also maintain the number and function of stem cells in order to meet the needs of seeding cells and afford novel strategies for prevention and treatment of age-related diseases.