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| miR-124-3p经Wnt通路因子Axin1调控骨髓间充质干细胞的研究 |
| The study of miR-124-3p regulating bone marrow mesenchymal stem cells via Wnt pathway factor Axin1 |
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| DOI：10.3969/j.issn.1006.7108.2022.03.001 |
| 中文关键词: 绝经后骨质疏松 骨髓间充质干细胞 miR-124-3p Axin1 |
| 英文关键词:postmenopausal osteoporosis BMSCs miR-124-3p Axin1 |
| 基金项目:广东省医学科学技术研究基金（A2020377） |
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| 中文摘要: |
| 目的 探讨miR-124-3p经Wnt通路因子A(Axin1)对骨髓间充质干细胞（BMSCs）的分子靶向机制及调节BMSCs的成骨分化进而参与绝经后骨质疏松（PMOP）的形成。方法 收集20例股骨骨折需要手术并扩髓或暴露髓腔的患者，10例绝经后骨质疏松妇女（PMOP组）和10例非骨质疏松的绝经后妇女（对照组），术中取骨髓组织3~5 mL，建立体外BMSCs细胞模型，用流式细胞仪检测BMSCs标志物，将BMSCs诱导分化为成骨细胞，RT-qPCR法检测miR-124-3p的表达。双荧光素酶报告基因系统检测miR-124-3p结合位点A-Axin1。构建miR-124-3p过表达载体，miR-124-3p过表达转染PMOP-BMSCs同时更换成骨诱导培养基进行成骨诱导培养。诱导第7、14、21 天 分别收集细胞进行检测碱性磷酸酯酶染色（ALP）和茜素红染色。成骨诱导第7天，RT-qPCR检测各组BMSCs中Runx2、Osterix靶因子Axin1的表达。Western Blotting检测miR-124-3p转染BMSCs后的表达与Axin1关系。结果 ①RT-qPCR法检测miR-124-3p的表达，PMOP组低于对照组。②miR-124-3p过表达转染、成骨诱导，随着诱导时间的延长，细胞培养液中ALP水平逐渐上升，茜素红染色可见结节沉积逐增加。③RT-qPCR检测成骨诱导第7天，Runx2和Osterix的表达，BMSC+成骨分化液组明显高于BMSC组，BMSC+过表达miR-124-3p组较其对照组明显上升；Axin1的表达，BMSC+成骨分化液组明显低于BMSC组，BMSC+过表达miR-124-3p组较其对照组显降低。④荧光素酶报告基因实验证明了miR-124-3p与Axin1的可结合性。⑤Western Blotting检测BMSCs，miR-124-3p过表达组Axin1表达显著下降，而miR-124-3p抑制组Axin1表达显著升高。结论 miR-124-3p可通过Wnt通路靶点Axin1调节BMSCs的成骨分化进而参与PMOP的形成。 |
| 英文摘要: |
| Objective To investigate the molecular targeting mechanism of miR-124-3p regulation of BMSCs osteogenic differentiation via factor A (Axin1) by bone marrow mesenchymal stem cells (BMSCs), and its participation in the development of postmenopausal osteoporosis (PMOP). Methods Twenty patients with femoral fractures requiring surgery or exposing the medullary cavity were collected, including 10 postmenopausal women with osteoporosis (PMOP group) and 10 non-osteoporosis postmenopausal women (control group). Bone marrow tissue (3-5 ml) was extracted during the operation. A bone mesenchymal stem cells (BMSCs) cell model was established. Surface marker antigens of BMSCs were detected using flow cytometry. BMSCs were induced to differentiate into osteoblasts. The expression of miR-124-3p was detected using RT-qPCR. The binding sites of A-Axin1 to miR-124-3p was detected with double luciferase reporter gene system. Overexpression vector of miR-124-3p was constructed. miR-124-3p was overexpressed and transfected to PMOP-BMSCs. Osteogenic induction medium was used for osteogenic induction culture. On 7d, 14d, 21d of induction, the cells were collected. Alkaline phosphatase (ALP) and Alizarin red staining were detected. On day 7, RT-qPCR was used to detect Runx2, Osterix, and target factor Axin1 in each group. The relationship between miR-124-3p expression after miR-124-3p transfected in BMSCs and Axin1 was detected using Western blotting. Results ①RT-qPCR was used to detect miR-124-3p expression. The expression in PMOP group was lower than that in control group. ②In cultures of overexpressed transfection and induced osteogenesis of miR-124-3p, the level of alkaline phosphatase and nodules of Alizarin red positive staining increased gradually with the extension of osteogenic induction time. ③RT-qPCR was used to detect the expressions of Runx2 and Osterix on day 7 of osteogenic induction. The expressions of Runx2 and Osterix in BMSC+ osteogenic differentiation group were significantly higher than those in BMSC group, and they were significantly higher in BMSC+ overexpression of miR-124-3p group than in control group. The expression of Axin1 in BMSC+ osteogenic differentiation group was significantly lower than in BMSC group. It was significantly lower in BMSC+ overexpression of miR-124-3p group than in control group. ④Luciferase reporter gene experiments demonstrated the binding of miR-124-3p to Axin1. ⑤Western blotting detection in BMSCs showed that the expression of Axin1 in miR-124-3p overexpressed group decreased significantly while the expression of Axin1 in miR-124-3p inhibitory group increased significantly. Conclusion miR-124-3p regulates BMSCs osteogenic differentiation and participates in PMOP formation via Wnt pathway target factor Axin1. |
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