LncRNA HAGLR absorbing miR-214-3p promotes BMP2 expression and improves tibial fractures

Abstract

Objective: To determine whether long-chain non-coding RNA (lncRNA) HAGLR can regulate BMP2 by absorbing microRNA-214-3p (miR-214-3p), and to explore its role and mechanism in tibial fracture (TF) healing.

Methods: The HAGLR, miR-214-3p, and BMP2 expression levels in TF and in adjacent normal tissues were measured using quantitative real-time polymerase chain reaction (qRT-PCR). MC3T3-E1 osteoblasts were used to construct the in vitro model. HAGLR was localized subcellularly through RNA-fluorescence in situ hybridization (FISH). A dual-luciferase report experiment confirmed that miR-214-3p has a targeted relationship with HAGLR and BMP2. It was then divided into a HAGLR over-expression group, an miR-214-3p mimic group, a HAGLR+miR-214-3p mimic group, an sh-HAGLR group, a BMP over-expression group, an sh-HAGLR+over-expression BMP2 group, and a negative control group. The proliferation and apoptosis of the MC3T3-E1 osteoblasts were examined using MTT assays and flow cytometry. A TF model was established in male C57BL/6J mice. The serum alkaline phosphatase (ALP) and osteoprotegerin (OPG) levels in the sham group, the TF group, and the TF group that were injected with HAGLR were compared using ELISA. Hematoxylin-eosin (HE) staining was used to confirm the fracture healing in the mouse model.

Results: Compared with the adjacent normal tissues in the TF patients, the HAGLR and BMP2 expressions decreased but the miR-214-3p expressions increased in the TF tissues (P<0.05). HAGLR, an endogenous sponge, absorbed the miR-214-3p, and the BMP2 expression was directly regulated by miR-214-3p. HAGLR increased the proliferative activity of the osteoblasts and decreased the apoptosis rate. The over-expression of miR-214-3p partly reversed the effect of HAGLR on the cells, decreased the proliferative activity, and increased the apoptosis rate (all P<0.05). The sh-HAGLR decreased the proliferative activity and increased the apoptosis rate. But after the over-expression of BMP2, the proliferative activity of the cells was higher, and the apoptosis rate was lower than it was in the sh-HAGLR group (all P<0.05). The over-expression of HAGLR can up-regulate the ALP and OPG levels in mouse models (P<0.05).

Conclusion: lncRNA HAGLR can regulate BMP2 to play a protective role in TF by absorbing miR-214-3p, and it is related to promoting the osteoblast proliferation, inhibiting apoptosis, and up-regulating the serum ALP and OPG levels to accelerate bone healing.

Keywords: Tibial fractures; apoptosis; lncRNA HAGLR; osteoblasts; proliferation.

Figure 1

The relative expression of lncRNA HAGLR. A: The expression of HAGLR in the TF tissues (n=42) and adjacent normal tissues (n=42) is measured using qRT-PCR; B: A diagnostic value curve (AUC=0.8798, 95% CI: 0.8076-0.9520, P<0.0001).

Figure 2

The effect of HAGLR on the proliferation and apoptosis of the osteoblasts. A: The expression of HAGLR after the transfection; B: The proliferation of the MC3T3-E1 cells was measured using MTT assays; C: The apoptosis of the MC3T3-E1 cells was measured using flow cytometry. Compared with the pcDNA3.1 group, *P<0.05; compared with the sh-HAGLR-NC group, #P<0.05; n=3.

Figure 3

HAGLR absorbed miR-214-3p to regulate its expression in osteoblasts. A: The prediction from the LncSNP website (http://210.46.80.146/lincsnp/search.php); B: Fluorescence in situ hybridization (400×); C: Dual-luciferase reporter assay; D: qRT-PCR is used to determine the expression of miR-214-3p in the adjacent healthy tissues (n=42) and the TF tissues (n=42); E: Correlation analysis; F: The expression of miR-214-3p in the cells after the HAGLR intervention is measured using qRT-PCR; compared with the mimic-NC group, ▲P<0.05; compared with the pcDNA3.1 group, *P<0.05; compared with the sh-HAGLR-NC group, #P<0.05; n=3.

Figure 4

The role of HAGLR in the osteoblastic growth. A: The expression of miR-214-3p in the cells was measured using qRT-PCR; B: MTT assays are used to measure the cell proliferation; C: The apoptosis was measured using flow cytometry. Compared with the mimic-NC group, %P<0.05; compared with the miR-214-3p mimic+pcDNA3.1 group, &P<0.05; n=3.

Figure 5

lncRNA HAGLR regulated the expression of its direct target BMP2 in TF by absorbing miR-214-3p. A: From the StarBase database (http://starbase.sysu.edu.cn/index.php), we found that BMP2 is a direct target of miR-214-3p; B: The interaction between BMP2 and the first 15 risk genes associated with increased fracture probability; C: A dual-luciferase report experiment verifies the relationship between BMP2 and miR-214-3p; D: The BMP2 expressions in the TF tissues (n=42) and the adjacent healthy tissues (n=42) were tested using qRT-PCR; E: The BMP2 mRNA level in the MC3T3-E1 cells was measured using qRT-PCR; F: The BMP2 protein level in the MC3T3-E1 cells was measured using WB; Compared with the mimic-NC and the BMP2-WT co-transfection group, ΔP<0.05; compared with the mimic-NC group, %P<0.05; compared with the miR-214-3p mimic+pcDNA3.1 group, &P<0.05; n=3.

Figure 6

The role of BMP2 in osteoblast growth. A: MTT assays were used to measure the cell proliferation. B: The apoptosis was measured using flow cytometry. Compared with the sh-HAGLR-NC group, ^P<0.05; Compared with the sh-HAGLR-NC+pcDNA3.1 group, @P<0.05; n=3.

Figure 7

The role of HAGLR in promoting bone healing in the TF mice. A: The HE staining observation of the mouse tissue morphology (200×); (a-c) are the results of the high power microscopic observations of the sham group (n=5), TF (n=5) and the TF+pcDNA3.1-HAGLR group (n=5; 400×); B: The serum ALP levels in each group (n=5); C: The serum OPG levels in the mice in each group (n=5). Compared with the sham group, ●P<0.05; compared with the TF group, ■P<0.05; n=3.

Figure 8

The mechanism by which LncRNA HAGLR absorbs miR-214-3p to promote the BMP2 expression to improve tibial fractures.