The Key Laboratory of Pathobiology, Ministry of Education, College of Basic Medical Sciences, Jilin University, 126 Xin Min Street, Changchun, Jilin Province, People's Republic of China.
The First Hospital, and Institute of Immunology, Jilin University, Changchun, 130021, China.
Stem Cell Res Ther. 2021 May 6;12(1):271. doi: 10.1186/s13287-021-02339-0.
Expansion-mediated replicative senescence and age-related natural senescence have adverse effects on mesenchymal stem cell (MSC) regenerative capability and functionality, thus severely impairing the extensive applications of MSC-based therapies. Emerging evidences suggest that microRNA-34a (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.
MiR-34a expression in MSCs was evaluated utilizing RT-qPCR. The functional effects of miR-34a exerting on MSC senescence were investigated via gene manipulation. Relevant gene and protein expression levels were analyzed by RT-qPCR and western blot. Luciferase reporter assays were applied to confirm that Nampt is a direct target of miR-34a. The underlying regulatory mechanism of miR-34a targeting Nampt in MSC senescence was further explored by measuring intracellular NAD content, NAD/NADH ratio and Sirt1 activity.
In contrast to Nampt expression, miR-34a expression incremented in senescent MSCs. 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 upregulated 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. In addition, 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.
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, carrying great implications for optimal strategies for MSC therapeutic applications.
扩增介导的复制性衰老和与年龄相关的自然衰老对间充质干细胞（MSC）的再生能力和功能有不利影响，从而严重损害了基于 MSC 的治疗的广泛应用。新出现的证据表明，miR-34a（miR-34a）已涉及 MSC 衰老过程；然而，miR-34a 影响 MSC 衰老的分子机制在很大程度上仍未确定。
利用 RT-qPCR 评估 MSC 中 miR-34a 的表达。通过基因操作研究 miR-34a 对 MSC 衰老的功能影响。通过 RT-qPCR 和 Western blot 分析相关基因和蛋白表达水平。应用荧光素酶报告实验证实 Nampt 是 miR-34a 的直接靶标。通过测量细胞内 NAD 含量、NAD/NADH 比和 Sirt1 活性，进一步探讨 miR-34a 靶向 MSC 衰老中 Nampt 的调节机制。
与 Nampt 表达相反，衰老 MSC 中 miR-34a 的表达增加。年轻 MSC 中 miR-34a 的过表达导致衰老相关特征，表现为衰老样形态、细胞增殖延长、成骨分化能力下降、衰老相关-β-半乳糖苷酶活性升高以及衰老相关因子的表达水平上调。相反，复制性衰老和自然衰老 MSC 中 miR-34a 的抑制作用导致衰老相关表型特征减少。我们确定了 Nampt 是 miR-34a 的直接靶基因。此外，miR-34a 补充导致 Nampt 表达水平、NAD 含量、NAD/NADH 比和 Sirt1 活性显著降低，而抗 miR-34a 处理则产生相反的效果。此外，通过 Nampt 过表达共处理明显挽救了 miR-34a 介导的 MSC 衰老。
本研究通过靶向 Nampt 并进一步通过 NAD-Sirt1 途径介导，确定了 miR-34a 在 MSC 复制性衰老和自然衰老中发挥的重要作用，这对优化 MSC 治疗应用的策略具有重要意义。
