Effect of microRNA-21 on hypoxia-inducible factor-1α in orthodontic tooth movement and human periodontal ligament cells under hypoxia

Abstract

Orthodontic tooth movement can lead to temporary hypoxia of periodontal tissues. Periodontal ligament cells (PDLCs) react to hypoxia, releasing various biological factors to promote periodontal tissue reconstruction. Hypoxia-inducible factor-1α (HIF-1α) is one of the most sensitive factors involved in the response to hypoxia. HIF-1α has been identified to be involved in osteogenic and osteoclast differentiation in vitro; however, few studies have investigated the expression of HIF-1α in the periodontal ligament (PDL) during orthodontic movement in vivo. In a previous study, microRNA-21 (miR-21) was demonstrated to be highly expressed in a rat model of orthodontic tooth movement. Additionally, miR-21 can increase the expression of HIF-1α in certain tumor cell types and is involved in tumor bioactivities. In the present study, HIF-1α exhibited expression patterns in a similar way to miR-21 in PDL samples from a rat model of orthodontic tooth movement, with expression initially increased and followed by a decrease over time. Furthermore, human PDLCs were exposed to a hypoxic environment in vitro, which induced significant upregulation of HIF-1α and miR-21 expression. Furthermore, miR-21 mimics increased HIF-1α expression and promoted osteogenic differentiation, indicated by upregulated expression of the osteogenic markers osteopontin, runt-related gene-2 and alkaline phosphatase. miR-21 inhibitors suppressed HIF-1α expression and downregulated the osteogenic markers. In conclusion, the results revealed that miR-21 has a positive effect on HIF-1α expression in PDLCs under hypoxia and has important roles in osteogenic differentiation during orthodontic tooth movement. These findings provide a theoretical basis by which to promote tissue reconstruction during orthodontic tooth movement.

Keywords: hypoxia; hypoxia-inducible factor-1α; microRNA-21; orthodontic tooth movement; periodontal ligament cells.

Figure 1.

Immunohistochemical staining (magnification, ×200). (A) HIF-1α protein expression in rat PDL on the tension side in the experimental group. HIF-1α expression was initially increased and then decreased, with a maximum increase indicated at day 7. (B) HIF-1α protein expression in rat PDL on the tension side in the control group. (C) HIF-1α protein expression in rat PDL on the pressure side in the experimental group. HIF-1α expression initially increased and then decreased, with a maximum increase at day 7. (D) HIF-1α protein expression in rat PDL on the pressure side in the control group. (E) Optical density of HIF-1α protein expression levels in tooth movement by immunohistochemical staining. Significantly high expression was identified at day 7 on tension sides and at day 5, 7 and 14 on pressure sides (**P<0.01 vs. the control group). PDL, periodontal ligament; HIF-1α, hypoxia-inducible factor-1α; d, day; a, alveolar; r, root.

Figure 2.

Expression of HIF-1α and miR-21 in periodontal ligament cells under hypoxia. (A) Expression of HIF-1α mRNA was rapidly and significantly increased under hypoxia at 6 h compared with the normoxia group, then the expression of HIF-1α mRNA slowly decreased. (B) miR-21 was significantly increased in periodontal ligament cells when exposed to hypoxia and remained significantly higher under hypoxia from 6 to 48 h compared with the normoxia group. (C and D) HIF-1α protein exhibited significantly higher expression at 6 and 12 h, and also decreased slowly (*P<0.05, **P<0.01 vs. the 48 h group). HIF-1α, hypoxia-inducible factor-1α; miR, microRNA.

Figure 3.

Effect of miR-21 on HIF-1α and osteogenic differentiation. (A) Transfection efficiency of miR-21 mimics. RT-qPCR confirmed that miR-21 mimics increased the expression of miR-21. (B) Transfection efficiency of miR-21 inhibitors. RT-qPCR confirmed that miR-21 inhibitors significantly decreased the expression of miR-21. (C) miR-21 mimics significantly increased the expression of HIF-1α by 3.9-fold, and miR-21 inhibitors decreased the expression of HIF-1α by 0.7-fold. (D) miR-21 mimics upregulated HIF-1α protein by 1.5-fold, and significantly increased the protein expression of osteogenic markers-OPN by 1.7-fold, RUNX-2 by 2.1-fold and ALP by 1.2-fold. (E) miR-21 inhibitors suppressed HIF-1α protein by 0.1-fold, and significantly decreased the protein expression of RUNX-2 by 0.6-fold and ALP by 0.7-fold. No significant difference was identified in OPN expression between the control and miR-21 inhibitors group (*P<0.05, **P<0.01 vs. the control group). miR, microRNA; HIF-1α, hypoxia-inducible factor-1α; OPN, osteopontin; RUNX-2, runt-related gene-2; ALP, alkaline phosphatase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.