Chen Jinlong, Yang Yishan
Department of Orthopedics, Zhangzhou Affiliated Hospital of Fujian Medical University Zhangzhou 363000, Fujian Province, China.
Am J Transl Res. 2021 Oct 15;13(10):11065-11080. eCollection 2021.
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 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 (<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 <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 <0.05). The over-expression of HAGLR can up-regulate the ALP and OPG levels in mouse models (<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.
目的：确定长链非编码RNA（lncRNA）HAGLR是否可通过吸附微小RNA-214-3p（miR-214-3p）来调控骨形态发生蛋白2（BMP2），并探讨其在胫骨骨折（TF）愈合中的作用及机制。 方法：采用定量实时聚合酶链反应（qRT-PCR）检测TF及其相邻正常组织中HAGLR、miR-214-3p和BMP2的表达水平。使用MC3T3-E1成骨细胞构建模型。通过RNA荧光原位杂交（FISH）对HAGLR进行亚细胞定位。双荧光素酶报告实验证实miR-214-3p与HAGLR和BMP2存在靶向关系。然后将其分为HAGLR过表达组、miR-214-3p模拟物组、HAGLR+miR-214-3p模拟物组、sh-HAGLR组、BMP过表达组、sh-HAGLR+BMP2过表达组和阴性对照组。采用MTT法和流式细胞术检测MC3T3-E1成骨细胞的增殖和凋亡情况。在雄性C57BL/6J小鼠中建立TF模型。采用酶联免疫吸附测定（ELISA）比较假手术组、TF组和注射HAGLR的TF组小鼠血清碱性磷酸酶（ALP）和骨保护素（OPG）水平。采用苏木精-伊红（HE）染色确认小鼠模型中的骨折愈合情况。 结果：与TF患者的相邻正常组织相比，TF组织中HAGLR和BMP2表达降低，但miR-214-3p表达升高（<0.05）。HAGLR作为内源性海绵吸附miR-214-3p，且BMP2表达受miR-214-3p直接调控。HAGLR增加了成骨细胞的增殖活性并降低了凋亡率。miR-214-3p的过表达部分逆转了HAGLR对细胞的作用，降低了增殖活性并增加了凋亡率（均<0.05）。sh-HAGLR降低了增殖活性并增加了凋亡率。但BMP2过表达后，细胞的增殖活性更高，凋亡率低于sh-HAGLR组（均<0.05）。HAGLR的过表达可上调小鼠模型中的ALP和OPG水平（<0.05）。 结论：lncRNA HAGLR可通过吸附miR-214-3p调控BMP2，在TF中发挥保护作用，这与促进成骨细胞增殖、抑制凋亡以及上调血清ALP和OPG水平以加速骨愈合有关。
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