Journal of Prevention and Treatment for Stomatological Diseases ›› 2020, Vol. 28 ›› Issue (9): 569-574.doi: 10.12016/j.issn.2096-1456.2020.09.004

• Basic Study • Previous Articles     Next Articles

Effect of miR-21 on the proliferation and osteogenic differentiation of human periodontal ligament stem cells

MA Lingzhi(),SHI Jiaozhuang,GE Wenbin,ZHANG Kun,YU Bing,LIU Yali()   

  1. Department of Orthodontics, the Affiliated Stomatological Hospital of Kunming Medical University, Yunnan 650101, China
  • Received:2019-12-25 Revised:2020-02-03 Online:2020-09-20 Published:2020-08-24
  • Contact: Yali LIU E-mail:422385890@qq.com;123460772@qq.com

Abstract:

Objective To explore the effect of miR-21 on human periodontal ligament stem cells (PDLSCs) proliferation and osteogenesis and to provide a theoretical basis for the stem cell treatment of periodontitis.Methods hPDLSCs were isolated and cultured with the enzymatic tissue block method, and surface molecules (CD34, CD45, CD90 and CD105) were detected by flow cytometry. An miR-21 mimics (pre-miR-21) and inhibitor (anti-miR-21) were transfected into hPDLSCs by Lipofectamine 2000. The experiment groups: mimics-NC group, mimics group, inhibitor group, and inhibitor-NC group. The transfection efficiency of miR-21 was determined by qRT-PCR. Proliferation was detected by CCK-8 and flow cytometry. The osteogenic differentiation ability of hPDLSCs was determined by alizarin red staining. Western blot was used to detect the protein expression of osteogenic related genes: Runx2.Results The mRNA expression of miR-21in the mimics group was significantly higher than that in the mimics-NC group; additionally, the expression in the inhibitor group was significantly weaker than that in the inhibitor-NC group (P < 0.05). hPDLSCs proliferation and the S phase cell ratio in the mimics group were stronger than those in the mimics-NC group(P < 0.05); those in the inhibitor group were weaker than those in the inhibitor-NC group (P < 0.05). After alizarin red staining, the mimics group was found to have more mineralized modules than mimics-NC group, and the inhibitor group had fewer than that in the inhibitor-NC group. Runx2 protein expression in the mimics group was higher than that in the mimics-NC group (P <0.05), and expression was lower in the inhibitor group than in the inhibitor-NC group (P < 0.05).Conclusion miR-21can promote the proliferation and osteogenesic differentiation of hPDLSCs.

Key words: microRNA, miR-21, periodontal tissue regeneration, human periodontal stem cells, proliferation, osteogenesis differentiation, mineralized module, runt-related transcription factor-2

CLC Number: 

  • R78

Table 1

Primer sequences"

Primer Sequence
miR-21-F 5′-GTGCAGGGTCCGAGGT-3′
miR-21-R 5′-GCCGCTAGCTTATCAGACTGATGT-3′
pre-miR-21-NC-F 5′-UUUGUACUACACAAAAGUACUG-3′
pre-miR-21-NC-R 5′-AAACAUGAUGUGUUUUCAUGAC-3′
anti-miR-21-NC 5′-CAGUACUUUUGUGUAGUACAAA -3′
U6-F 5′-CTCGCTTCGGCAGCACA-3′
U6-R 5′-AACGCTTCACGAATTTGCGT-3′

Figure 1

Primary culture of hPDLSCs a: on the 6th day, primary hPDLSCs were seen around the tissue block (× 100); b: after passage, hPDLSCs showed typical vortex-like growth (× 100); hPDLSCs: human periodontal ligament stem cells"

Figure 2

Surface molecules of hPDLSCs were measured by flow cytometry a: hPDLSCs showed low expression of hematopoietic stem cell surface markers: CD34(1.51%); b: hPDLSCs showed low expression of hematopoietic stem cell surface markers: CD45(1.01%); c: hPDLSCs showed high expression of mesenchymal surface markers: CD90(94.16%); d: hPDLSCs showed high expression of mesenchymal surface markers: CD105(98.33%); hPDLSCs: human periodontal ligament stem cells"

Figure 3

Relative expression of miR-21 in transfected hPDLSCs detected by qRT-RCR **: P<0.01"

Figure 4

Proliferation of hPDLSCs after overexpression and inhibition of miR-21 In the first 24 h, there was no significant difference in the cell proliferation level of each group. From 24 h to 96 h, the proliferation in the mimics group was significantly promoted and that in the inhibitor group was significantly inhibited"

Figure 5

Cell cycle of hPDLSCsafter overexpression and inhibition of miR-21 a: mimics group; b: mimics-NC; c: inhibitor; d: inhibitor-NC"

Figure 6

Images of mineralized nodules by alizarin red staining after overexpression or inhibition of miR-21 in the 4 groups a: mimics-NC; b: mimics; c: inhibitor-NC; d: inhibitor (× 100)"

Figure 7

Protein expression of Runx2 after overexpression or inhibition of miR-21 The expression level of the osteogenic related protein Runx2 in the mimics group was significantly higher than that in the mimics-NC group, amd the expression in the inhibitor group was lower than that in the inhibitor-NC group. mimics vs. mimics-NC*, P<0.05. inhibitor vs. inhibitor-NC*, P<0.05"

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