Let‐7b downgrades CCND1 to repress osteogenic proliferation and differentiation of MC3T3‐E1 cells: An implication in osteoporosis

Abstract

Abstract The aim of this study was to reveal the effect of let‐7b on osteoporosis (OP). Synthetic let‐7b mimics or inhibitors were transfected into MC3T3‐E1 cells. The expression of let‐7b in MC3T3‐E1 and its effect on cell viability, apoptosis, and the apoptosis‐related proteins (Bcl‐2, Bax, and cleaved caspase‐9) were tested by CCK‐8 assay, flow cytometry and Western blot, severally. The osteogenic differentiation markers (Runx2 and Osterix) and Wnt/β‐catenin pathway related markers (β‐catenin and C‐myc) were detected by qRT‐PCR and Western blot. The relationships between let‐7b and cyclin D1 (CCND1) were confirmed by luciferase reporter assay. The differentiation and mineralization of MC3T3‐E1 cells were analyzed by alkaline phosphatase (ALP) activity assay and alizarin red staining. The outcomes indicated that overexpression/ablation of let‐7b repressed/facilitated MC3T3‐E1 cell viability and accelerated/suppressed MC3T3‐E1 cell apoptosis. Besides, a remarkable decrease/augment of Bcl‐2 protein expression and the distinct fortify/reduction of Bax and cleaved caspase‐9 expression levels were observed in let‐7b mimics/inhibitors group in MC3T3‐E1 cells. Moreover, we discovered that let‐7b overexpression/ablation retrained/facilitated the mRNA and protein expression of Runx2 and Osterix. It was confirmed that CCND1 was a downstream target of let‐7b and was negatively modulated by let‐7b. In addition, high‐expression/deficiency of let‐7b inhibited/increased the expression levels of β‐catenin and C‐myc in MC3T3‐E1 cells. Taken together, our study revealed that let‐7b overexpression/depletion repressed/accelerated MC3T3‐E1 cell proliferation, differentiation, and mineralization while promoted/suppressed MC3T3‐E1 cell apoptosis through targeting CCND1, which might be adjusted by Wnt/β‐catenin pathway. Our findings might offer a basis for developing novel targets for OP treatment.

Keywords

• CCND1
• let‐7b
• osteoblast differentiation
• osteoporosis
• Wnt/β‐catenin signaling pathway