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Journal of Peking University (Health Sciences) ›› 2026, Vol. 58 ›› Issue (1): 10-21. doi: 10.19723/j.issn.1671-167X.2026.01.002

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miR-488-5p promotes osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells and enhances neuralized bone regeneration

Liting ZENG1, Kaiyuan CHENG1,2, Zhongning LIU1, Jian LI1, Jingwen YANG1,*(), Ting JIANG1,*()   

  1. 1. Department of Prosthodontics, Peking University School and Hospital of Stomatology & National Center for Stomatology & National Clinical Research Center for Oral Diseases & National Engineering Research Center of Oral Biomaterials and Digital Medical Devices, Beijing 100081, China
    2. Department Ⅰ of Prosthodontics, Tianjin Stomatological Hospital, School of Medicine, Nankai University, Tianjin Key Laboratory of Oral and Maxillofacial Function Reconstruction, Tianjin 300041, China
  • Received:2025-10-10 Online:2026-02-18 Published:2025-12-10
  • Contact: Jingwen YANG, Ting JIANG
  • Supported by:
    the National Natural Science Foundation of China(82170928); the National Natural Science Foundation of China(82201022)

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Abstract:

Objective: To investigate the role of microRNA miR-488-5p, which showed increased expression after the disconnection of the inferior alveolar nerve, in promoting the osteogenic and neurogenic differentiation of rat bone marrow mesenchymal stem cells (rBMSCs), as well as its effect on promoting the neuralized tissue engineered bone regeneration. Methods: rBMSCs were subjected to in vitro neural or osteogenic differentiation induction cultures. The expression levels of miR-488-5p at different time points (days 0, 2, 4, and 7) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). miR-488-5p overexpression or low expression in rBMSCs was achieved by transfection with miR-488-5p mimics or inhibitors. Four groups, the miR-488-5p mimics, the miR-488-5p inhibitor, and their respective negative controls (NC), were established to investigate the effects of miR-488-5p on the neural differentiation and osteogenic differentiation of rBMSCs.A 5 mm diameter, full-thickness circular critical bone defect was created in the rat calvaria. The rats were treated with light-cured gelatin methacryloyl (GelMA) seeded with rBMSCs. The rats were divided into four groups: ①BLANK group: GelMA; ②BMSCs group: GelMA + rBMSCs; ③NC-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics NC; and ④miR-488-5p-BMSCs group: GelMA + rBMSCs transfected with miR-488-5p mimics. Specimens were obtained 4 and 8 weeks after surgery, and micro-CT was performed to measure and analyze bone mineral density (BMD), bone volume/total volume (BV/TV), bone surface area/total volume (BS/TV) and trabecular number (Tb.N). The effects of neuralized tissue engineering bone formation in the defect area were assessed using Hematoxylin-Eosin (HE) staining, Masson staining, and tissue immunofluorescence staining of the nerve-specific protein soluble protein-100 (S100). Results: As rBMSCs progressed toward neural or osteogenic differentiation, miR-488-5p expression increased significantly from day 0 to day 7. Regarding neural differentiation, the mimics group showed increased expression of neural-related genes and proteins compared with the mimics NC group, while the opposite result was observed in the inhibitor group. As for osteogenic differentiation, the mimics group showed increased expression of osteogenic genes and proteins, more intense alkaline phosphatase (ALP) and alizarin red staining (ARS) staining, and enhanced ALP activity compared with the mimics NC group, while the opposite result was observed in the inhibitor group. 4 and 8 weeks after critical calvarial defect construction in rats, the BLANK group had the least amount of new bone formation, while the BMSCs group and the NC-BMSCs group had similar and intermediate amounts of new bone formation. The miR-488-5p-BMSCs group had the most new bone formation. At 4 weeks, the BMD [(0.63±0.05) g/cm3 vs. (0.51±0.03) g/cm3], BV/TV (33.17%±6.43% vs. 18.11%±1.52%), BS/TV [(3.43±0.69) /mm vs. (2.46±0.20) /mm], and Tb.N [(0.92±0.21) /mm vs.(0.59±0.07) /mm] in the miR-488-5p-BMSCs group were significantly higher than those in the NC-BMSCs group. At 8 weeks, the BMD [(0.80±0.04) g/cm3 vs. (0.68±0.04) g/cm3], BV/TV (56.69%±6.22% vs. 42.36%±3.86%), and the number of S100-labeled nerve cells around the new bone (46.33±4.04 vs. 26.00±3.61) in the miR-488-5p-BMSCs group was also significantly higher than that in the NC-BMSCs group. Conclusion: miR-488-5p promoted the osteogenic and neurogenic differentiation of rBMSCs and promoted the formation of neuralized tissue-engineered bone in rat calvarial defects.

Key words: miR-488-5p, Bone marrow mesenchymal stem cells, Osteogenic differentiation, Neurogenic differentiation, Neuro-bone tissue engineering

CLC Number: 

  • R68

Table 1

Primer sequences used for qRT-PCR"

Gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′)
U6 CGCTTCGGCAGCACATATAC CGAATTTGCGTGTCATCCTT
miR-488-5p GGCACCCAGATAATGGCAC AGTGCAGGGTCCGAGGTATT
Gapdh AGGTCGGTGTGAACGGATTT GAACTTGCCGTGGGTAGAGT
*Map2 ACAGCAACAAGTGGTGAATCAG GGAGGATGGAGGAAGGTCTTG
*Ng2 TTCTCACACAGAGGAGCCC CACTCAAGCTCTGGCTGCT
*Tubb3 CAGATGCTGGCCATTCAGAGTAAG TGTTGCCGATGAAGGTGGAC
*Ngf TGCCAAGGACGCAGCTTTC TGAAGTTTAGTCCAGTGGGCTTCAG
*Nes TGAACAAGAGACCCAACAAACAC TTCCAAGAGGCTTCGGTAACT
*S100 CTGTCAAGAACCTGCTCCGA AGTGGGCATGGAACACATTGA
#Runx2 GCCTTCAAGGTTGTAGCCCT TGAACCTGGCCACTTGGTTT
#Alp CATGGTGAGTGACACGGACA CCATGACGTGGGGGATGTAG
#Ocn CCGTTTAGGGCATGTGTTGC TTTCGAGGCAGAGAGAGGGA
#Bmp2 CGGGAACAAATGCAGGAAGC AAGGACATTCCCCATGGCAG
#Col1a1 CCCCAGCCGCAAAGAGTCTA CAGCTGACTTCAGGGATGTCTTC
#Opn CCAGCCAAGGACCAACTACA AGTGTTTGCTGTAATGCGCC

Figure 1

miR-488-5p promotes neurogenic differentiation of rBMSCs A, mRNA expression levels of neurogenic-related genes Nes, S100, Map2, Ng2, Tubb3, Ngf on day 12 of neurogenic differentiation in the NM and PM groups as detected by qRT-PCR; B, immunofluorescence detection of Tubb3 in the NM and PM groups; C, miR-488-5p expression levels on days 0, 2, 4 and 7 of neurogenic differentiation as detected by qRT-PCR; D, mRNA expression levels of neurogenic-related genes Nes, S100, Map2, Ng2, Tubb3, Ngf on day 12 of neurogenic differentiation as detected by qRT-PCR; E, protein expression levels of Tubb3 and Gap43 as detected by Western blot; F, immunofluorescence detection of Tubb3 and quantification of Tubb3 fluorescence intensity. ns, not significant. * P < 0.05, * * P < 0.01, * * * P < 0.001. PM, proliferation medium; NM, neurogenic medium; Mimics NC, negative control for miR-488-5p mimics; Inhibitor NC, negative control for miR-488-5p inhibitor; Nes, nestin; S100, soluble protein-100; Map2, microtubule-associated protein 2; Ng2, neural/glial antigen 2; Tubb3, class Ⅲ β-tubulin; Ngf, nerve growth factor; Gap43, growth associated protein 43;DAPI, 4', 6-diamidino-2-phenylindole; rBMSCs, rat bone marrow mesenchymal stem cells; qRT-PCR, quantitative real-time polymerase chain reaction."

Figure 2

miR-488-5p promotes osteogenic differentiation of rBMSCs A, mRNA expression levels of osteogenic-related genes Runx2, Alp, Col1a1, Opn, Bmp2, Ocn on day 7 of osteogenic differentiation in the OM and PM groups as detected by qRT-PCR; B, ALP staining on day 7 of osteogenic differentiation in the OM and PM groups; C, miR-488-5p expression levels on days 0, 2, 4 and 7 of osteogenic differentiation as detected by qRT-PCR; D, mRNA expression levels of osteogenic-related genes Runx2, Alp, Col1a1, Opn, Bmp2, Ocn on day 7 of osteogenic differentiation as detected by qRT-PCR; E, protein expression levels of Runx2 and Osx as detected by Western blot; F, ALP staining on day 7 and ARS staining on day 21 of osteogenic differentiation; G, ALP activity on day 7 of osteogenic differentiation; H, quantification of mineralization by measuring ARS absorbance at 562 nm. ns, not significant. * P < 0.05, * * P < 0.01, * * * P < 0.001. PM, proliferation medium; OM, osteogenic medium; d, day; Mimics NC, negative control for miR-488-5p mimics; Inhibitor NC, negative control for miR-488-5p inhibitor; Runx2, Runt-related transcription factor 2; ALP, alkaline phosphatase; Ocn, Osteocalcin; Bmp2, bone morphogenetic protein 2; Col1a1, collagen type Ⅰ alpha 1 chain; Opn, osteopontin; ARS, alizarin red S; rBMSCs, rat bone marrow mesenchymal stem cells; qRT-PCR, quantitative real-time polymerase chain reaction."

Figure 3

micro-CT imaging analysis of rat calvarial defects at 4 and 8 weeks after operation A, 3D reconstructed images after micro-CT scanning showing bone defect areas at both 4 and 8 weeks after operation; B, quantitative comparisons of BMD, BV/TV, BS/TV, Tb.N at both 4 and 8 weeks after operation. ns, not significant. * P < 0.05, * * P < 0.01, * * * P < 0.001. W, weeks after operation. BLANK, the control group treated with GelMA only; BMSCs, the group treated with GelMA and normal rBMSCs; NC-BMSCs, the group treated with GelMA and rBMSCs transfected with negative control (NC) for miR-488-5p mimics; miR-488-5p-BMSCs, the group treated with GelMA and rBMSCs transfected with miR-488-5p mimics; BMD, bone mineral density; BV/TV, bone volume/total volume; BS/TV, bone surface area/total volume; Tb.N, trabecular number; 3D, 3-dimensional; BMSCs, bone marrow mesenchymal stem cells."

Figure 4

Histomorphological analysis of rat calvarial defects at 4 and 8 weeks after operation A, HE staining and Masson staining; B, quantitative measurement of new bone area in Masson staining at 8 weeks after operation; C, immunofluorescence detection of S100 protein; D, analysis of the number of S100-positive cells in immunofluorescence staining at 8 weeks after operation. HE, Hematoxylin-Eosin; NB, new bone; S100, soluble protein-100; DAPI, 4', 6-diamidino-2-phenvlindole; W, weeks after operation; BLANK, the control group treated with GelMA only; BMSCs, the group treated with GelMA and normal rBMSCs; NC-BMSCs, the group treated with GelMA and rBMSCs transfected with negative control (NC) for miR-488-5p mimics; miR-488-5p-BMSCs, the group treated with GelMA and rBMSCs transfected with miR-488-5p mimics; BMSCs, bone marrow mesenchymal stem cells. ns, not significant; * P < 0.05, * * P < 0.01."

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