Metformin-Induced MicroRNA-34a-3p Downregulation Alleviates Senescence in Human Dental Pulp Stem Cells by Targeting CAB39 through the AMPK/mTOR Signaling Pathway.

Shuo Zhang,Pengyan Qiao,Lingling E,Chuanjie Li,Rongjian Lu,Hongchen Liu,Feifan Wang,Rong Zhang,Xiaocao Ma,Federica Facchin

doi:10.1155/2021/6616240

Abstract

Dental pulp stem cells (DPSCs) are ideal seed cells for the regeneration of dental tissues. However, DPSC senescence restricts its clinical applications. Metformin (Met), a common prescription drug for type 2 diabetes, is thought to influence the aging process. This study is aimed at determining the effects of metformin on DPSC senescence. Young and aging DPSCs were isolated from freshly extracted human teeth. Flow cytometry confirmed that DPSCs expressed characteristic surface antigen markers of mesenchymal stem cells (MSCs). Cell Counting Kit-8 (CCK-8) assay showed that a concentration of 100 μM metformin produced the highest increase in the proliferation of DPSCs. Metformin inhibited senescence in DPSCs as evidenced by senescence-associated β-galactosidase (SA-β-gal) staining and the expression levels of senescence-associated proteins. Additionally, metformin significantly suppressed microRNA-34a-3p (miR-34a-3p) expression, elevated calcium-binding protein 39 (CAB39) expression, and activated the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) signaling pathway. Dual-luciferase reporter assay confirmed that CAB39 is a direct target for miR-34a-3p. Furthermore, transfection of miR-34a-3p mimics promoted the senescence of DPSCs, while metformin treatment or Lenti-CAB39 transfection inhibited cellular senescence. In conclusion, these results indicated that metformin could alleviate the senescence of DPSCs by downregulating miR-34a-3p and upregulating CAB39 through the AMPK/mTOR signaling pathway. This study elucidates on the inhibitory effect of metformin on DPSC senescence and its potential as a therapeutic target for senescence treatment.

Highlights

Stem cells are used for restoring injured tissues because of their self-renewal, multipotent differentiation, and paracrine signaling properties
This study is aimed at determining the effects of metformin on miR-34a-3p and investigating the molecular mechanisms underlying the function of miR-34a-3p, focusing on its target gene calcium-binding protein 39 (CAB39) and the AMPK/mammalian target of rapamycin (mTOR) signaling pathway, in regulating the senescence of Dental pulp stem cells (DPSCs)
Flow cytometry demonstrated that both young and aging DPSCs positively expressed human mesenchymal stem cells (MSCs) surface markers CD44 (99.9%, 99.9%), CD90 (99.9%, 100%), and CD105 (99.4%, 99.5%), while negatively expressing hematopoietic cell markers CD11b (0%, 0.61%), CD14 (1.33%, 0.82%), CD34 (0.12%, 0.035%), and CD45 (0.49%, 0.87%) (Figure 1(c)). These results indicated that DPSCs conformed to the characteristics of MSCs

Summary

Introduction

Stem cells are used for restoring injured tissues because of their self-renewal, multipotent differentiation, and paracrine signaling properties. Stem cells derived from dental tissues, such as dental pulp, tooth germ, apical papilla, and periodontal ligament, are abundant in number and accessible. They are considered attractive candidates for tissue engineering and regenerative medicine [1]. Dental pulp stem cells (DPSCs) were the first human dental mesenchymal stem cells (MSCs) to be isolated from pulp tissue [2] These cells can undergo chondrogenic, osteogenic, adipogenic, neurogenic, and odontogenic differentiation [3] and can, be used for dental tissue regenerative therapy in pulp revascularization, dentine formation, and periodontal regeneration [4].

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