Abstract
Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells' (MSCs') biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.
Highlights
Periodontal disease is a chronic inflammatory disease, which usually leads to periodontal tissue destruction [1]
We mainly examined whether and how hsa-let-7b affects the osteogenic differentiation of periodontal ligament stem cells (PDLSCs)
PDLSCs were obtained from the middle third of root surfaces of premolars successfully (Figures 1(a) and 1(b))
Summary
Periodontal disease is a chronic inflammatory disease, which usually leads to periodontal tissue destruction (including gingival bleeding, periodontal pocket formation and inflammation, attachment loss, and alveolar bone absorption) [1]. Studies have shown that PDLSCs have multidirectional differentiation potential and strong self-renewal ability These cells can differentiate into bone, adipose, and cartilage tissue [5]. Collagen triple helix repeat containing 1 (CTHRC1) gene is located on human chromosome 8q22.3, encoding a 28 kDa secretory protein [15] It is highly conserved in chordate animals, while there is no homolog found in lower species such as flies and worms. Up to now, there are few studies on whether and how CTHRC1 affects the osteogenic differentiation PDLSCs. Since miRNA hsa-let-7b plays a significant role in the committed differentiation of stem cells, and CTHRC1 is forecasted to be the target gene of hsa-let-7b by bioinformatics analysis, we hypothesized that the let-7b may clarify a certain key mechanism for the directed differentiation of stem cells by regulating CTHRC1. We mainly examined whether and how hsa-let-7b affects the osteogenic differentiation of PDLSCs
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