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. 2022 Jan;10(2):37.
doi: 10.21037/atm-21-6101.

The regulatory roles of miR-26a in the development of fracture and osteoblasts

Jilong Zou  1 , Jiabing Sun  1 , Hongjun Chen  1 , Xinming Fan  1 , Zhenrui Qiu  1 , Yuan Li  1 , Jianhui Shi  2   3
Affiliations

The regulatory roles of miR-26a in the development of fracture and osteoblasts

Jilong Zou et al. Ann Transl Med. 2022 Jan.

Abstract

Background: MicroRNAs (miRNAs) play a vital role in the bone development and bone regeneration. In this study, we investigated the effects of miR-26a in osteoblasts and fractures.

Methods: Human osteoblasts were cultured and used for analysis. To identify differential miRNAs in blood samples from patients with fractures and healthy controls, quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed. Human osteoblasts were transfected with miR-26a mimics, miR-26a inhibitor, or their corresponding negative controls (NCs), respectively. MTT assay was performed to identify the effects of miR-26a on the cell viability of osteoblasts. EdU staining was applied to detect the proliferation of osteoblasts. Trypan blue staining was utilized to analyze the effects of miR-26a on the cell death of osteoblasts. Terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling (TUNEL) staining was used to detect apoptotic osteoblasts. Alizarin red S (ARS) staining and qRT-PCR analysis were utilized to measure the mineralized nodule formation to evaluate the bone formation of osteoblasts. Dual luciferase reporter assay and western blot analysis were performed to detect the relationship between miR-26a and its target gene.

Results: The results of qRT-PCR analysis identified miR-26a as our miRNA of interest and indicated that miR-26a was significantly decreased in patients with fractures. Overexpression of miR-26a significantly increased the cell viability and proliferation of osteoblasts. An increase in miR-26a reduced the cell death and apoptosis of osteoblasts, and promoted the osteoblastic activity and mineralized nodule formation. Dual luciferase reporter assay, qRT-PCR and western blot analysis showed that miR-26a could negatively regulate the expression of phosphatase and tensin homolog (PTEN).

Conclusions: MiR-26a promoted new bone regeneration via regulating the functions of osteoblasts by targeting its target gene PTEN. Therefore, we propose that targeting miR-26a may be a novel therapeutic method for bone regeneration and treating fractures.

Keywords: MicroRNAs (miRNAs); fracture; mineralized nodule formation; osteoblast.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-21-6101/coif). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The expression of miR-26a was decreased in patients with fractures. (A) The expression levels of five miRNAs in patients with fractures were detected by qRT-PCR analysis. (B) The expression level of miR-26a in patients with fractures before and after treatment. Values are the mean ± SD of three independent experiments. *P<0.05, **P<0.01, ***P<0.001 versus control. MiRNAs, microRNAs; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation.
Figure 2
Figure 2
MiR-26a elevated the cell viability and proliferation ability of osteoblasts. (A) The expression of miR-26a in osteoblasts after transfection of miR-26a mimics and mimics NC. (B) The expression of miR-26a in osteoblasts after transfection of the miR-26a inhibitor and inhibitor NC. (C,D) MTT assay was applied to detect the cell viability of osteoblasts after transfection with miR-26a. (E,F) The EdU staining was performed to assess the roles of miR-26a in the proliferation of osteoblasts (40×). Values are the mean ± SD of three independent experiments. ***P<0.001, ****P<0.0001 versus control. NC, negative control; MTT, 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di- phenytetrazoliumromide; SD, standard deviation; OD, optical density; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.
Figure 3
Figure 3
MiR-26a inhibited the cell death and apoptosis of osteoblasts. (A,B) The trypan blue assay was utilized to determine the effects of miR-26a on the cell death of osteoblasts, and the statistical results are shown. (C,D) The apoptosis of osteoblasts after transfection with miR-26a mimics or miR-26a inhibitor was measured by TUNEL staining (40×). Values are the mean ± SD of three independent experiments (n=3). **P<0.01, ***P<0.001 versus control. TUNEL, terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling; SD, standard deviation; NC, negative control; DAPI, 2-(4-Amidinophenyl)-6-indolecarbamidine dihydrochloride.
Figure 4
Figure 4
Overexpression of miR-26a promoted the activity of osteoblasts. (A-C) To detect the role of miR-26a in the expression of osteoblast-related genes, qRT-PCR analysis was performed. (D,E) ARS staining was applied to detect the roles of miR-26a in the mineralized nodule formation of osteoblasts (10×). Values are the mean ± SD of three independent experiments (n=3). **P<0.01, ***P<0.001, ****P<0.0001 versus control. qRT-PCR, quantitative real-time polymerase chain reaction; ARS, Alizarin red S; SD, standard deviation; ALP, alkaline phosphatase; BMP4, bone morphogenetic protein 4; OCN, osteocalcin; NC, negative control.
Figure 5
Figure 5
MiR-26a directly targets PTEN. (A) Illustration of the binding sites of PTEN and miR-26a. (B) The roles of miR-26a in the luciferase activities of pmirGLO-PTEN-WT and pmirGLO-PTEN-MUT were quantified by luciferase reporter assay. (C) The expression of PTEN in miR-26a overexpressing or depleting cells by qRT-PCR analysis. (D,E) The pictures and statistical diagram of the western blot was shown. Values are the mean ± SD of three independent experiments (n=3). ***P<0.001, ****P<0.0001 versus control. PTEN, phosphatase and tensin homolog; qRT-PCR, quantitative real-time polymerase chain reaction; SD, standard deviation; NC, negative control.
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