Department of Orthopedics, Mindong Hospital Affiliated to Fujian Medical University, Fu'an, China.
Eur Rev Med Pharmacol Sci. 2019 Jul;23(14):6011-6017. doi: 10.26355/eurrev_201907_18410.
To study the mechanism of micro ribonucleic acid (miR)-140-3p participating in the regulation of fracture healing in rats.
A total of 50 male Sprague-Dawley (SD) rats were randomly divided into five groups, namely, group A [phosphate-buffered saline (PBS)] (n=10), group B (miR-140-3p mimics) (n=10), group C [ mimics negative control (NC)] (n=10), group D [antisense oligonucleotide (ASO)-miR-140-3p] (n=10), and group E (ASO NC) (n=10). A rat model of fracture was established on all the rats through the operation. From the successful establishment of the model, the rats in group A were intraperitoneally injected with 50 μL PBS (2 nmol) once a week for 6 weeks, and those in group B, C, D, and E were injected with equivalent volume of miR-140-3p mimics, mimics NC, ASO-miR-140-3p, and ASO NC, respectively, once a week since the successful establishment of model for 6 weeks. The fracture healing in the rats was evaluated via imaging. Meanwhile, Real Time-Polymerase Chain Reaction (RT-PCR) was applied to detect the expression of miR-140-3p in the five groups. Wnt and β-catenin expressions in the five groups were detected by means of Western blotting (WB). Alkaline phosphatase (ALP) and its quantized statistical value in the five groups were detected through immunohistochemical staining.
The expression of miR-140-3p was stimulated in miR-140-3p mimics group and inhibited in ASO-miR-140-3p group. The detection of the miR-140-3p expression level in the five groups via RT-PCR showed that miR-140-3p mimics group had a remarkably higher miR-140-3p expression than the other four groups. The differences were statistically significant (p<0.05). The WB assay verified that the Wnt and β-catenin expressions in miR-140-3p mimics group were notably higher than those in control groups, and there were statistically significant differences (p<0.05). Compared with those in the groups injected with PBS, ASO miR-140-3p, mimics NC, and ASO NC, there were evidently more callus tissues, better healed and more blurred fracture lines, as well as no translocation and looseness of internal fixation, in the group injected with miR-140-3p mimics, suggesting that the stimulation of the miR-140-3p expression promotes the fracture healing in the rats. The results of immunohistochemical staining indicated that the number of ALP-positive osteoblasts in the rats in miR-140-3p mimics group was increased markedly in comparison with that in the remaining groups (p<0.05), implying that the differentiation of osteoblasts in the rats was affected in miR-140-3p mimics group.
The overexpressed miR-140-3p in the rats with fracture can promote fracture healing by activating the Wnt signaling pathway.
研究微小 RNA-140-3p（miR-140-3p）参与调控大鼠骨折愈合的机制。
将 50 只雄性 Sprague-Dawley（SD）大鼠随机分为 5 组，即 A 组[磷酸盐缓冲液（PBS）]（n=10）、B 组（miR-140-3p 模拟物）（n=10）、C 组（miR-140-3p 模拟物阴性对照（NC））（n=10）、D 组[反义寡核苷酸（ASO）-miR-140-3p]（n=10）和 E 组（ASO NC）（n=10）。通过手术在所有大鼠中建立骨折模型。从模型成功建立起，A 组大鼠每周腹腔内注射 50 μL PBS（2 nmol）一次，共 6 周，B、C、D 和 E 组大鼠在模型成功建立后每周分别注射等量的 miR-140-3p 模拟物、模拟物 NC、ASO-miR-140-3p 和 ASO NC，共 6 周。通过影像学评估大鼠骨折愈合情况。同时，采用实时聚合酶链反应（RT-PCR）检测五组大鼠 miR-140-3p 的表达。通过 Western blot（WB）检测五组大鼠 Wnt 和 β-catenin 的表达。通过免疫组织化学染色检测五组大鼠碱性磷酸酶（ALP）及其量化统计值。
miR-140-3p 模拟物组 miR-140-3p 表达受刺激，ASO-miR-140-3p 组 miR-140-3p 表达受抑制。五组大鼠 miR-140-3p 表达水平的 RT-PCR 检测结果显示，miR-140-3p 模拟物组 miR-140-3p 表达明显高于其他四组，差异有统计学意义（p<0.05）。WB 检测证实，miR-140-3p 模拟物组 Wnt 和 β-catenin 的表达明显高于对照组，差异有统计学意义（p<0.05）。与注射 PBS、ASO-miR-140-3p、模拟物 NC 和 ASO NC 的大鼠相比，注射 miR-140-3p 模拟物的大鼠骨痂组织明显增多，愈合较好，骨折线更模糊，内固定无移位和松动，表明 miR-140-3p 表达的刺激促进了大鼠骨折愈合。免疫组织化学染色结果表明，与其他组相比，miR-140-3p 模拟物组大鼠中 ALP 阳性成骨细胞数量明显增加（p<0.05），提示 miR-140-3p 模拟物组大鼠成骨细胞分化受到影响。
骨折大鼠中过表达的 miR-140-3p 通过激活 Wnt 信号通路促进骨折愈合。
