miR-24-3p靶向BMP8B对成骨细胞功能影响的实验研究

doi:10.3969/j.issn.1006-6233.2023.01.0011

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摘要 目的: 探讨miR-24-3p与骨形态发生蛋白8B(BMP8B)的靶向关系,并分析二者对成骨细胞功能的影响。方法: 体外分离培养人牙周膜干细胞(hPDLSCs),并将其诱导分化为成骨细胞,采用碱性磷酸酶(ALP)染色法、茜素红染色法鉴定成骨细胞;将成骨细胞分为空白对照组、阴性对照组(inhibitor-NC组)、沉默miR-24-3p表达组(miR-24-3p-inhibitor组)、共转染组(miR-24-3p-inhibitor+BMP8B-siRNA组)。采用实时荧光定量PCR法检测miR-24-3p、BMP8B mRNA水平;CCK-8法、流式细胞仪分别检测各组成骨细胞增殖、凋亡情况;采用ALP活性检测试剂盒检测ALP活性;免疫印迹法检测各组成骨细胞BMP8B蛋白、功能活性相关蛋白(PICP、BGP、OPN)水平。应用TargetScan数据库预测miR-24-3p与BMP8B靶向关系并用双荧光素酶报告基因实验验证。结果: 与空白对照组、inhibitor-NC组比较,miR-24-3p-inhibitor组成骨细胞增殖抑制率、凋亡率降低,ALP活性及PICP、BGP、OPN表达升高(P<0.05);与miR-24-3p-inhibitor组比较,miR-24-3p-inhibitor+BMP8B-siRNA组成骨细胞增殖抑制率、凋亡率升高,ALP活性及PICP、BGP、OPN表达降低(P<0.05)。双荧光素酶报告基因实验证实miR-24-3p靶向调控BMP8B。结论: 抑制miR-24-3p可能通过靶向上调BMP8B表达促进成骨细胞增殖及分化,并抑制细胞凋亡。

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关键词 ： miR-24-3p, 骨形态发生蛋白8B, 成骨细胞, 增殖, 凋亡

Abstract：Objective: To investigate the targeting relationship between miR-24-3p and bone morphogenetic protein 8B (BMP8B), and to analyze their effects on osteoblast function. Methods: Human periodontal ligament stem cells (hPDLSCs) were isolated and cultured in vitro and induced to differentiate into osteoblasts. The osteoblasts were identified by alkaline phosphatase (ALP) staining and alizarin red staining; the osteoblasts were divided into blank control group, negative control group (inhibitor-NC group), silencing miR-24-3p expression group (miR-24-3p-inhibitor group), and co-transfection group (miR-24-3p-inhibitor +BMP8B-siRNA group). Real-time fluorescent quantitative PCR method was used to detect the levels of miR-24-3p and BMP8B mRNA; CCK-8 method and flow cytometer were used to detect the proliferation and apoptosis of bone cells in each component; ALP activity detection kit was used to detect ALP activity; Western blotting was used to detect the levels of BMP8B protein and functional activity-related proteins (PICP, BGP, OPN) in each component of bone cells. The TargetScan database and the dual luciferase reporter gene experiment were used to predict and verify the targeting relationship between miR-24-3p and BMP8B respectively. Results: Compared with the blank control group and inhibitor-NC group, the osteoblast proliferation inhibition rate and apoptosis rate in the miR-24-3p-inhibitor group were decreased, ALP activity and expression of PICP, BGP, OPN were increased (P<0.05); compared with miR-24-3p-inhibitor group, the osteoblast proliferation inhibition rate and apoptosis rate in the miR-24-3p-inhibitor+BMP8B-siRNA group were increased, ALP activity and expression of PICP, BGP, OPN were decreased (P<0.05). The dual luciferase reporter gene experiment confirmed that miR-24-3p targeted regulation of BMP8B. Conclusion: Inhibition of miR-24-3p may promote the proliferation and differentiation of osteoblasts and inhibit cell apoptosis by targeting up-regulation of BMP8B expression.

Key words： miR-24-3p Bone morphogenetic protein 8B Osteoblasts Proliferation Apoptosis

基金资助:河北省卫生健康委医学科学研究课题,(编号:20210670)

引用本文:

王建琪, 李召宝, 吴帅楠, 郭香君, 曹素敏, 林秀雅, 褚亚辉. miR-24-3p靶向BMP8B对成骨细胞功能影响的实验研究[J]. 河北医学, 2023, 29(1): 59-65.
WANG jianqi, LI Zhaobao, WU Shuanan, et al. Experimental Study on the Effect of miR-24-3p Targeting BMP8B on the Function of Osteoblasts. HeBei Med, 2023, 29(1): 59-65.

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http://www.hbyxzzs.cn/CN/10.3969/j.issn.1006-6233.2023.01.0011 或 http://www.hbyxzzs.cn/CN/Y2023/V29/I1/59

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