| 赵娜,王亚妹,李宏,等.微 RNA-758通过调控细胞周期素 D1促进人牙周膜干细胞的骨生成[J].安徽医药,2026,30(8):1631. |
| 微 RNA-758通过调控细胞周期素 D1促进人牙周膜干细胞的骨生成 |
| miR-758 promotes osteogenesis of human periodontal ligament stem cells by regulating cyclin D1 |
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| DOI：10.3969/j.issn.1009-6469.2026.08.031 |
| 中文关键词: 骨生成 微 RNA-758 细胞周期素 D1 牙周膜干细胞 成骨分化 成骨相关转录因子 骨钙素 多潜能干细胞 |
| 英文关键词: Osteogenesis MiR-758 Cyclin D1 Periodontal ligament stem cells Osteogenic differentiation Osterix Osteo-calcin Pluripotent stem cells |
| 基金项目:河北省重点研发计划项目( 20220228241D) |
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| 中文摘要: |
| 目的探究微 RNA(miR)-758通过细胞周期素 D1(CCND1)对人牙周膜干细胞( PDLSCs)成骨分化的影响。方法 2023年 3月至 2024年 4月从基因综合数据库( GEO)获得成骨诱导与非诱导 PDLSCs之间差异表达的 miRNA。该研究采用基于细胞转染方式进行分组，具体分组及处理如下: miR-758 mimic组、 mimic-NC组、 miR-758-inhibitor组、 inhibitor-NC组， mimic-NC+oe-NC组、 miR-758+oe-NC组、 miR-758+oe-CCND1组。应用碱性磷酸酶( ALP)染色和茜素红 S染色分别评价 ALP活性和矿化程度。通过实时荧光定量逆转录聚合酶链反应( qRT-PCR)检测 PDLSCs中 miR-758、CCND1的表达；蛋白质印迹法检测成骨相关转录因子( OSX)、骨钙素和 Runt相关转录因子 2(RUNX2)的相对表达水平。通过功能获得和丧失实验研究 miR-758的作用。此外，通过双萤光素酶报告基因实验评估 miR-758与 CCND1的调控关系。结果成骨诱导后的 PDLSCs第 1、7、14、21天 miR-758表达分别为 1.00±0.09、2.13±0.11、3.79±0.21、4.58±0.36，与非诱导 PDLSCs相比， miR-758在经历成骨诱导的 PDLSCs中的表达水平显著增加( F=161.22，P<0.001)； inhibitor-NC组、 miR-758-inhibitor组、 mimic-NC组、 miR-758-mimic组 miR-758表达分别为 1.00±0.09、0.19±0.04、1.00±0.10、4.32±0.24，ALP活性分别为 0.14±0.02、0.03±0.01、0.11±0.01、0.28±0.03(F=522.81、86.93，均 P <0.001)，过表达 miR-758促进了 PDLSCs分化过程中 ALP活性、 RUNX2、骨钙素和 OSX的表达，而敲低 miR-758则抑制了 PDLSCs分化过程中 ALP活性、 RUNX2、骨钙素和 OSX的表达。其次，茜素红染色证实过表达 miR-758促进成骨细胞矿化程度，而敲低表达 miR-758降低成骨细胞矿化程度。此外，进一步双萤光素酶报告实验结果证实 miR-758通过靶向 CCND1的 3'-UTR以调控 CCND1的表达。过表达 miR-758导致 CCND1的表达水平降低，敲低 miR-758导致 CCND1的表达水平提高。最后，拯救实验结果显示过表达 miR-758促进 PDLSCs成骨分化，而进一步过表达 CCND1则部分消除了这一作用。结论 miR-758/ CCND1调控网络在调控 PDLSCs成骨分化过程中发挥促进作用。 |
| 英文摘要: |
| Objective To investigate the effect of microRNA (miR)-758 on the osteogenic differentiation of human periodontal liga-ment stem cells (PDLSCs) through cyclin D1 (CCND1).Methods From March 2023 to April 2024, differentially expressed miRNAs between osteogenesis-induced and non-induced PDLSCs were obtained from the Gene Expression Omnibus (GEO) database. In this study, cell-based transfection was used for grouping, and the specific grouping and treatment were as follows: miR-758 mimic group, mimic-NC group, miR-758-inhibitor group, inhibitor-NC group, mimic-NC+oe-NC group, miR-758+oe-NC group, and miR-758+oe-CCND1 group. Alkaline phosphatase (ALP) staining and alizarin red S staining were used to evaluate ALP activity and mineralization,respectively. The expressions of miR-758 and CCND1 in PDLSCs were detected by real-time quantitative reverse transeriptase poly-merase chain reaction (qRT-PCR), and the expressions of osterix (OSX), osteocalcin (OCN) and runt-related transcription factor 2 (RUNX2) were detected by Western blotting. The effects of miR-758 were investigated through gain-of-function and loss-of-function ex-periments. In addition, the regulatory relationship between miR-758 and CCND1 was evaluated by dual luciferase reporter assays.Re. sults The expression levels of miR-758 in PDLSCs at days 1, 7, 14, and 21 after osteogenic induction were 1.00±0.09, 2.13±0.11,3.79±0.21 and 4.58±0.36, respectively. Compared with non-induced PDLSCs, the expression level of miR-758 was significantly in-creased in osteogenically induced PDLSCs (F=161.22, P<0.001);The miR-758 expression levels in the inhibitor-NC, miR-758-inhibi-tor, mimic-NC, and miR-758-mimic groups were 1.00±0.09, 0.19±0.04, 1.00±0.10 and 4.32±0.24, respectively, while the correspond-ing ALP activities were 0.14±0.02, 0.03±0.01, 0.11±0.01 and 0.28±0.03. All differences were statistically significant (F=522.81 for miR-758 expression; F=86.93 for ALP activity; P<0.001 for both comparisons). Overexpression of miR-758 promoted the expression of ALP, RUNX2, OCN and OSX during the differentiation of PDLSCs, while knockdown of miR-758 inhibited the expression of ALP activ-ity, RUNX2, OCN and OSX during the differentiation of PDLSCs. Second, alizarin red staining confirmed that overexpression of miR-758 promoted the degree of osteoblast mineralization, whereas knockdown expression of miR-758 decreased the degree of osteoblast mineralization. In addition, further results of dual luciferase reporter assay confirmed that miR-758 regulates CCND1 expression by tar-geting the 3'-UTR of CCND1. Overexpression of miR-758 resulted in decreased expression levels of CCND1, and knockdown of miR-758 resulted in increased expression levels of CCND1. Finally, rescue experiments showed that overexpression of miR-758 promoted osteogenic differentiation of PDLSCs, while further overexpression of CCND1 partially eliminated this effect.Conclusion The miR-758/CCND1 regulatory network plays a promoting role in regulating the osteogenic differentiation of PDLSCs. |
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