MiR-34a Suppression Ameliorates Bone Marrow Mesenchymal Stem Cell Senescence by Targeting Nampt and Regulating NAD <sup>+</sup>-Sirt1 Pathway

Abstract

Background: Expansion-mediated replicative senescence and age-related natural senescence have adverse effects on MSC regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that miR-34a has been implicated in the process of MSC senescence; however, the molecular mechanisms with regard to how miR-34a influencing MSC senescence remain largely undetermined. Methods: We undertake a systematic study on the functional effects of miR-34a exerting on MSC senescence via gene manipulation. Mechanistically, bioinformatics analysis and dual-luciferase reporter gene assay were applied to decipher the interactions between miR-34a and its potential target Nampt. Findings: miR-34a overexpression in young MSCs resulted in senescence-associated characteristics as displayed by senescence-like morphology, prolonged cell proliferation, declined osteogenic differentiation potency, heightened senescence-associated-β- galactosidase activity, and up-regulated expression levels of the senescence-associated factors. Conversely, miR-34a suppression in replicative senescent and natural senescent MSCs contributed to diminished senescence-related phenotypic features. We identified Nampt as a direct target gene of miR-34a. Additionally, miR-34a repletion resulted in prominent reductions in Nampt expression levels, NAD+ content, NAD+/NADH ratio, and Sirt1 activity, whereas anti-miR-34a treatment exerted the opposite effects. Furthermore, miR-34a-mediated MSC senescence was evidently rescued following the co-treatment with Nampt overexpression. Interpretation: This study identifies a significant role of miR-34a playing in MSC replicative senescence and natural senescence via targeting Nampt and further mediating by NAD+-Sirt1 pathway, possibly carrying great implications for optimal strategies for MSC therapeutic applications. Funding Statement: This study was supported by projects of National Natural Science Foundation of China (Grant number: 81571370), Jilin Province Science and Technology Development Projects (Grant number: 20150414029GH), Science and Technology Projects of the Education Department of Jilin Province (Grant Number: JJKH20190007KJ) and the Fundamental Research Funds for Central Universities, JLU. Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: Experimental investigations involving animals were permitted by the Institutional Animal Care and the Ethic Committee of Jilin University (permit number: SYXK 2013-0005) and were conducted in compliance with the ethical standards and regulations, the Declaration of Helsinki and national and international guidelines.