scholarly journals Wnt16 signaling promotes osteoblast differentiation of periosteal derived cells in vitro and in vivo

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e10374
Author(s):  
Ying Jin ◽  
Xiaoyan Sun ◽  
Fang Pei ◽  
Zhihe Zhao ◽  
Jeremy Mao
Keyword(s):  
Bone Formation ◽  
Mrna Expression ◽  
Nuclear Translocation ◽  
Luciferase Activity ◽  
Fracture Repair ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Alizarin Red ◽  
Osteogenic Induction

Background Periosteum plays critical roles in de novo bone formation and fracture repair. Wnt16 has been regarded as a key regulator in periosteum bone formation. However, the role of Wnt16 in periosteum derived cells (PDCs) osteogenic differentiation remains unclear. The study goal is to uncover whether and how Wnt16 acts on the osteogenesis of PDCs. Methods We detected the variation of Wnt16 mRNA expression in PDCs, which were isolated from mouse femur and identified by flow cytometry, cultured in osteogenic medium for 14 days, then knocked down and over-expressed Wnt16 in PDCs to analysis its effects in osteogenesis. Further, we seeded PDCs (Wnt16 over-expressed/vector) in β-tricalcium phosphate cubes, and transplanted this complex into a critical size calvarial defect. Lastly, we used immunofluorescence, Topflash and NFAT luciferase reporter assay to study the possible downstream signaling pathway of Wnt16. Results Wnt16 mRNA expression showed an increasing trend in PDCs under osteogenic induction for 14 days. Wnt16 shRNA reduced mRNA expression of Runx2, collage type I (Col-1) and osteocalcin (OCN) after 7 days of osteogenic induction, as well as alizarin red staining intensity after 21days. Wnt16 also increased the mRNA expression of Runx2 and OCN and the protein production of Runx2 and Col-1 after 2 days of osteogenic stimulation. In the orthotopic transplantation assay, more bone volume, trabecula number and less trabecula space were found in Wnt16 over-expressed group. Besides, in the newly formed tissue Brdu positive area was smaller and Col-1 was larger in Wnt16 over-expressed group compared to the control group. Finally, Wnt16 upregulated CTNNB1/β-catenin expression and its nuclear translocation in PDCs, also increased Topflash reporter luciferase activity. By contrast, Wnt16 failed to increase NFAT reporter luciferase activity. Conclusion Together, Wnt16 plays a positive role in regulating PDCs osteogenesis, and Wnt16 may have a potential use in improving bone regeneration.

scholarly journals MiR-26b-5p Promotes Osteogenesis of Bone Mesenchymal Stem Cells via Suppressing FGF21

2021 ◽  
Author(s):  
Bin Wang ◽  
Zhenhui Li ◽  
Caiyuan Mai ◽  
Penglin Mou ◽  
Lei Pan
Keyword(s):  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Control Group ◽  
Osteogenic Differentiation Medium ◽  
Bone Mesenchymal Stem Cells ◽  
Alizarin Red ◽  
Osteogenic Induction ◽  
Direct Target ◽  
Inhibition Group ◽  
Fgf21 Expression

Abstract Introduction: It has been established that miR-26b-5p actively participate in the osteogenic differentiation of bone mesenchymal stem cells (BMSCs), which is of great value in osteoporosis treatment. Database showed that Fibroblast growth factor(FGF)21 is a potential binding site of miR-26b-5p. This study aimed to investigate the molecular osteogenic mechanisms of miR-26b-5p targeting FGF21 in postmenopausal osteoporosis (PMOP). Methods: 5ml of bone marrow was aspirated from the anterior superior iliac spine in 10 PMOP women during bone marrow puncture. BMSCs were used to establish an in vitro cell model, and BMSCs markers were analyzed by flow cytometry. miR-26b-5p and FGF21 were overexpressed for 48h, and then placed in an osteogenic induction medium for osteogenic induction culture, the expression of RNA was detect using RT-qPCR. Cells from miR-26b-5p group were collected on days 7, 14 and 21 of induction for ALP and alizarin red S staining. On day 7 of induction, RT-qPCR was used to measure Runx2, Osterix (Osx), and target gene FGF21 expression levels in each group. The dual-luciferase reporter gene system was used to verify that FGF21 was a direct target of miR-26b-5p. FGF21 was measured by western blotting in the miR-26b-5p overexpression group and in the miR-26b-5p inhibition group. Results: BMSCs were identified according with the antigenic characteristics. miR-26b-5p expression was significantly upregulated after the expression of miR-26b-5p mimics; however, FGF21 expression was downregulated after FGF21 mimics. After overexpression of miR-26b-5p, the alkaline phosphatase activity and nodules of alizarin red S in the culture medium gradually increased as the induction time increased. RT-qPCR showed that the expressions of master osteogenic factors Runx2 and Osx in the BMSC+ osteogenic differentiation medium group was significantly higher than in the BMSC group, the expressions of the factors in the BMSC+ miR-26b-5p overexpression group was significantly higher than in the control group. Target gene FGF21 expression was significantly lower in the BMSC+ osteogenic differentiation medium group than in the BMSC group, and was significantly lower in the BMSC+ miR-26b-5p overexpression group than in the control group. Luciferase reporter assays demonstrated that FGF21 was a direct target of miR-26b-5p. Finally, western blotting analysis showed that FGF21 expression was significantly downregulated in the miR-26b-5p overexpressed group and upregulated in the miR-26b-5p inhibition group. Conclusion: miR-26b-5p can regulate the osteogenic differentiation of BMSCs and participate in PMOP pathogenesis via suppressing FGF21. The present study provides the basis for further studies on PMOP.

MiR-144-3p Inhibits BMSC Proliferation and Osteogenic Differentiation Via Targeting FZD4 in Steroid-Associated Osteonecrosis

2020 ◽  
Vol 25 (45) ◽  
pp. 4806-4812 ◽  
Author(s):  
Zhibo Sun ◽  
Fei Wu ◽  
Yue Yang ◽  
Feng Liu ◽  
Fengbo Mo ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Nuclear Translocation ◽  
Bone Diseases ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Alizarin Red ◽  
Over Expression ◽  
Negative Effect ◽  
Proliferation Ability

Background: MicroRNAs have recently been recognized to be engaged in the development of bone diseases. Objective: This study was performed to elucidate the effects of miR-144-3p on proliferation and osteogenesis of mesenchymal stem cells (MSCs) from the patients with steroid-associated osteonecrosis (ONFH) and its related mechanism. Method: The expression level of miR-144-3p in the MSCs from the proximal femur of the patients was examined by Real-time PCR. The cell proliferation ability was assayed by MTT. The differentiation ability of MSCs was assayed by Alizarin Red S (ARS) staining. The interaction between miR-144-3p and frizzled4 (FZD4) was investigated by Real-time PCR, western blot and luciferase reporter assay. Results: ONFH samples had the obviously high expression of miR-144-3p compared to the control. MiR-144-3p had a negative effect on the proliferation and osteogenesis of MSCs. Via targeting FZD4, miR-144-3p decreased β-catenin nuclear translocation, the transcription of RUNX2 and COL1A1. Over-expression of FZD4 partially reversed miR-144-3p-induced decrease in the proliferation and osteogenesis of MSCs. Conclusion: MiR-144-3p might play an important role in the development of ONFH and might be used as a novel class of therapeutic targets for this disease.

scholarly journals MicroRNA-513b-5p targets COL1A1 and COL1A2 associated with the formation and rupture of intracranial aneurysm

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Zheng Zheng ◽  
Yan Chen ◽  
Yinzhou Wang ◽  
Yongkun Li ◽  
Qiong Cheng
Keyword(s):  
Cell Death ◽  
Intracranial Aneurysm ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Control Group ◽  
Type I ◽  
Mrna Levels ◽  
Reporter Assay ◽  
Over Expression ◽  
Tnf Α

AbstractCollagen-type I alpha 1 chain (COL1A1) and COL1A2 are abnormally expressed in intracranial aneurysm (IA), but their mechanism of action remains unclear. This study was performed to investigate the mechanism of COL1A1 and COL1A2 affecting the occurrence and rupture of IA. Quantitative real-time polymerase chain reaction was used to measure the expression of hsa-miR-513b-5p, COL1A1, COL1A2, TNF-α, IL-6, MMP2, MMP3, MMP9 and TIMP4 in patients with ruptured IA (RA) (n = 100), patients with un-ruptured IA (UA) (n = 100), and controls (n = 100). Then, human vascular smooth muscle cells (HASMCs) were cultured, and dual luciferase reporter assay was performed to analyse the targeting relationship between miR-513b-5p and COL1A1 or COL1A2. The effects of the miR-513b-5p mimic and inhibitor on the proliferation, apoptosis, and death of HASMC and the RIP1-RIP3-MLKL and matrix metalloproteinase pathways were also explored. The effect of silencing and over-expression of COL1A1 and COL1A2 on the role of miR-513b-5p were also evaluated. Finally, the effects of TNF-α on miR-513b-5p targeting COL1A1 and COL1A2 were tested. Compared with those in the control group, the serum mRNA levels of miR-513b-5p, IL-6 and TIMP4 were significantly decreased in the RA and UA groups, but COL1A1, COL1A2, TNF-α, IL-1β, MMP2, MMP3 and MMP9 were significantly increased (p < 0.05). Compared with those in the UA group, the expression of COL1A1, COL1A2, TNF-α, IL-1β and MMP9 was significantly up-regulated in the RA group (p < 0.05). Results from the luciferase reporter assay showed that COL1A1 and COL1A were the direct targets of miR-513b-5p. Further studies demonstrated that miR-513b-5p targeted COL1A1/2 to regulate the RIP1-RIP3-MLKL and MMP pathways, thereby enhancing cell death and apoptosis. Over-expression of COL1A1 or COL1A2, rather than silencing COL1A1/2, could improve the inhibitory effect of miR-513b-5p on cell activity by regulating the RIP1-RIP3-MLKL and MMP pathways. Furthermore, over-expression of miR-513b-5p and/or silencing COL1A1/2 inhibited the TNF-α-induced cell proliferation and enhanced the TNF-α-induced cell death and apoptosis. The mechanism may be related to the inhibition of collagen I and TIMP4 expression and promotion of the expression of RIP1, p-RIP1, p-RIP3, p-MLKL, MMP2 and MMP9. MiR-513b-5p targeted the inhibition of COL1A1/2 expression and affected HASMC viability and extracellular mechanism remodelling by regulating the RIP1-RIP3-MLKL and MMP pathways. This process might be involved in the formation and rupture of IA.

scholarly journals Transcutaneous CO2 application accelerates fracture repair in streptozotocin-induced type I diabetic rats

2020 ◽  
Vol 8 (2) ◽  
pp. e001129
Author(s):  
Takahiro Oda ◽  
Takahiro Niikura ◽  
Tomoaki Fukui ◽  
Keisuke Oe ◽  
Yu Kuroiwa ◽  
...  
Keyword(s):  
Endochondral Ossification ◽  
Callus Formation ◽  
Femoral Shaft ◽  
Shaft Fracture ◽  
Diabetic Rats ◽  
Fracture Repair ◽  
Female Rats ◽  
Control Group ◽  
Type I ◽  
Biomechanical Assessment

IntroductionDiabetes mellitus (DM) negatively affects fracture repair by inhibiting endochondral ossification, chondrogenesis, callus formation, and angiogenesis. We previously reported that transcutaneous CO2 application accelerates fracture repair by promoting endochondral ossification and angiogenesis. The present study aimed to determine whether CO2 treatment would promote fracture repair in cases with type I DM.Research design and methodsA closed femoral shaft fracture was induced in female rats with streptozotocin-induced type I DM. CO2 treatment was performed five times a week for the CO2 group. Sham treatment, where CO2 was replaced with air, was performed for the control group. Radiographic, histologic, genetic, and biomechanical measurements were taken at several time points.ResultsRadiographic assessment demonstrated that fracture repair was induced in the CO2 group. Histologically, accelerated endochondral ossification and capillary formation were observed in the CO2 group. Immunohistochemical assessment indicated that early postfracture proliferation of chondrocytes in callus was enhanced in the CO2 group. Genetic assessment results suggested that cartilage and bone formation, angiogenesis, and vasodilation were upregulated in the CO2 group. Biomechanical assessment revealed enhanced mechanical strength in the CO2 group.ConclusionsOur findings suggest that CO2 treatment accelerates fracture repair in type I DM rats. CO2 treatment could be an effective strategy for delayed fracture repair due to DM.

microRNA-4458 Regulates Insulin Resistance in Hepatic Cells by Targeting the Insulin-Like Growth Factor 1 Receptor

2021 ◽  
Vol 11 (9) ◽  
pp. 1812-1817
Author(s):  
Jingjing Zhou ◽  
Wenjuan Zhu ◽  
Zheng Mao ◽  
Zhen Li ◽  
Xiaoqin Li ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Mrna Expression ◽  
Glucose Uptake ◽  
Hepg2 Cell ◽  
Molecular Mechanisms ◽  
Cell Model ◽  
Serum Insulin ◽  
Luciferase Reporter ◽  
Control Group ◽  
Hepatic Cells

Background: The objective of the research was to investigate the roles of miR-4458 in the regulation of insulin resistance in hepatic cells and to explore the underlying molecular mechanisms. Methods: The blood samples were collected from the T2D patients and the health controls, and the liver tissues were collected from the DM and control rats. The relationship between IGF1R and miR-4458 was predicted by TargetScan and verified by the dual luciferase reporter gene system. qRT-PCR was used to measure the mRNA expression of miR-4458, IGF1R, G6Pase and PEPCK. The protein expression of IGF1R, p-AKT and AKT were measured by Western blot analysis. The rat insulin ELISA Kit and glucose Uptake Colorimteric Assay Kit were used to determine the level of serum insulin and the glucose uptake. Results: miR-4458 was high expressed in T2D patients. We predicted and verified that IGF1R was a direct target of miR-4458, and the mRNA expression of IGF1R was reduced in type 2 diabetes patients. We established the diabetes model (DM) and IR HepG2 cell model, and found that the blood glucose and serum insulin levels were significantly elevated in the DM group. miR-4458 expression was up-regulated, while the expression of IGF1R and p-AKT, and p-AKT/AKT ratio were reduced in the DM group and IR HepG2 cell model. miR-4458 inhibitor and IGF1R-siRNA significantly decreased the expression of miR-4458 and IGF1R respectively. In comparison with IR+inhibitor control group, miR-4458 inhibitor increased 2-DG6P content, IGF1R expression, p-AKT expression and p-AKT/AKT ratio, reduced the expression of G6Pase and PEPCK, and all the effects were reversed by down-regulating IGF1R. Conclusion: miR-4458 regulated the insulin resistance in hepatic cells by regulating the IGF1R/PI3K/AKT signal pathway, which will be a potential target for the treatment of diabetes.

scholarly journals Effect of Fibroblast Growth Factor 21 on the Expression of TLR4 and BAMBI Genes in Cholesterol-Treated Human Hepatic Stellate Cells

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Elham Shakerian ◽  
Narges Mohammad Taghvaei ◽  
Zohre Askari ◽  
Reza Afarin
Keyword(s):  
Growth Factor ◽  
Mrna Expression ◽  
Fibroblast Growth Factor ◽  
Hepatic Stellate Cells ◽  
Stellate Cells ◽  
Fibroblast Growth Factor 21 ◽  
Control Group ◽  
Type I ◽  
Fibroblast Growth ◽  
Human Hscs

Background: Activated hepatic stellate cells (HSCs) are the primary mediators in the progression of hepatic fibrosis. The activation of toll-like receptor 4 (TLR4) signaling leads to the downregulation of the transmembrane inhibitory transforming growth factor-beta (TGF-β) pseudoreceptor BMP and activin membrane-bound inhibitor (BAMBI) on HSCs. Fibroblast growth factor 21 (FGF21) is a natural secretory protein in the body with effects, such as the reduction of fat accumulation and oxidation of lipids; however; no study has investigated FGF21 ability to prevent the progression of liver fibrosis. Objectives: This study aimed to examine the beneficial effects of FGF21 to reduce cholesterol-activated human HSCs. Methods: The human HSCs were incubated in media containing different concentrations of cholesterol, including 25, 50, 75, 100, 125, and 150 μM, for 24 h and then incubated with FGF21 for 24 h. Total ribonucleic acids were extracted and reversely transcribed into complementary deoxyribonucleic acid. A quantitative real-time polymerase chain reaction was performed in this study. Results: The results showed that the messenger ribonucleic acid (mRNA) expression of TGF-β, collagen, type I, alpha 1 (collagen1α), and TLR4 genes increased significantly in the presence of cholesterol (75 and 100 μM), compared to that of the control group (* P < 0.05, ** P < 0.01, and *** P < 0.001); nevertheless, the mRNA expression of the BAMBI gene significantly reduced, compared to that of the control group (* P < 0.05). The FGF21 significantly reduced the mRNA expression of TGF-β, collagen1α, and TLR4 genes (# P < 0.05). The mRNA expression of the BAMBI gene significantly increased with FGF21 (# P < 0.05). Conclusions: It was concluded that the treatment with FGF21 reduces the cholesterol-activated HSCs by decreasing the mRNA expression of the TLR4, TGF-β, and collagen1α genes and increasing the mRNA expression of the BAMBI gene.

The Effect of Vitamin D and Its Metabolites on Fracture Repair in Chicks

1983 ◽  
Vol 65 (4) ◽  
pp. 429-436 ◽  
Author(s):  
S. Dekel ◽  
R. Salama ◽  
S. Edelstein
Keyword(s):  
Vitamin D ◽  
Bone Formation ◽  
Vitamin D3 ◽  
Fracture Repair ◽  
Control Group ◽  
Clinical Implications ◽  
Torsional Stress ◽  
Active Vitamin ◽  
Dihydroxyvitamin D3 ◽  
24,25 Dihydroxyvitamin D3

1. One-day-old chicks were depleted of vitamin D. At 3 weeks their right tibiae, and those of a control group given vitamin D3, were fractured and pinned. After fracture the controls were kept on vitamin D3. Another group was left vitamin D-deficient. The remaining depleted chicks, divided into four groups, were given vitamin D3, 24,25-dihydroxyvitamin D3 [24,25(OH)2D3], 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] or a combination of 24,25(OH)2D3 and 1,25(OH)2D3. 2. The callus obtained after 9 and 14 days was subjected to torsional stress. The callus of chicks given vitamin D continuously showed the greatest resistance, whereas that of vitamin D-deficient chicks showed the smallest resistance. Repletion with either vitamin D3 or its metabolites increased the strength of the callus. Repletion with the combination of 24,25(OH)2D3 and 1,25(OH)2D3 produced the most marked results, in that the callus was even stronger than that of chicks replete with vitamin D3. 3. It is concluded that 24,25(OH)2D3 is essential for bone formation in addition to the known active vitamin D metabolite 1,25(OH)2D3, and the possible clinical implications of these findings are discussed.

Losartan attenuates paraquat-induced pulmonary fibrosis in rats

2014 ◽  
Vol 34 (5) ◽  
pp. 497-505 ◽  
Author(s):  
F Guo ◽  
YB Sun ◽  
L Su ◽  
S Li ◽  
ZF Liu ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Lung Injury ◽  
Mrna Expression ◽  
Collagen Type ◽  
Collagen Type I ◽  
Control Group ◽  
Type I ◽  
Expression Levels ◽  
Relative Expression ◽  
Tgf Β1

Paraquat (PQ) is one of the most widely used herbicides in the world and can cause pulmonary fibrosis in the cases with intoxication. Losartan, an angiotensin II type 1 receptor antagonist, has beneficial effects on the treatment of fibrosis. The aim of this study was to examine the effect of losartan on pulmonary fibrosis in PQ-intoxicated rats. Adult male Sprague Dawley rats ( n = 32, 180–220 g) were randomly assigned to four groups: (i) control group; (ii) PQ group; (iii) PQ + losartan 7d group; and (iv) PQ + losartan 14d group. Losartan treatment (intragastrically (i.g.), 10 mg/kg) was performed for 7 and 14 days after a single i.g. dose of 40 mg/kg PQ. All rats were killed on the 16th day, and hematoxylin–eosin and Masson’s trichrome staining were used to examine lung injury and fibrosis. The levels of hydroxyproline and transforming growth factor β1 (TGF-β1), matrix metallopeptidase 9 (Mmp9), and tissue inhibitor of metalloproteinase 1 (TIMP-1) messenger RNA (mRNA) expression and relative expression levels of collagen type I and III were also detected. PQ caused a significant increase in hydroxyproline content, mRNA expression of TGF-β1, Mmp9, and TIMP-1, and relative expression levels of collagen type I and III (  p < 0.05), while losartan significantly decreased the amount of hydroxyproline and downregulated TGF-β1, Mmp9, and TIMP-1 mRNA and collagen type I and III expressions (  p < 0.05). Histological examination of PQ-treated rats showed lung injury and widespread inflammatory cell infiltration in the alveolar space and pulmonary fibrosis, while losartan could markedly reduce such damage and prevent pulmonary fibrosis. The results of this study indicated that losartan could reduce lung damage and prevent pulmonary fibrosis induced by PQ.

scholarly journals The Role and Mechanism of Exosomes from Umbilical Cord Mesenchymal Stem Cells in Inducing Osteogenesis and Preventing Osteoporosis

2021 ◽  
Vol 30 ◽  
pp. 096368972110574
Author(s):  
Ge Yahao ◽  
Wang Xinjia
Keyword(s):  
Stem Cell ◽  
Osteogenic Differentiation ◽  
Umbilical Cord ◽  
Cell Counting ◽  
Control Group ◽  
Human Umbilical Cord ◽  
Promoting Effect ◽  
Alizarin Red ◽  
Osteogenic Induction

Mesenchymal stem cell (MSC) exosomes promote tissue regeneration and repair, and thus might be used to treat many diseases; however, the influence of microenvironmental conditions on exosomes remains unclear. The present study aimed to analyze the effect of osteogenic induction on the functions of human umbilical cord MSC (HucMSC)-derived exosomes. Exosomes from standardized stem cell culture (Exo1) and osteogenic differentiation-exosomes (Exo2) were co-cultured with osteoblasts, separately. Cell counting kit-8 assays, alkaline phosphatase and alizarin red staining were used to observe the exosomes’ effects on osteoblast proliferation and differentiation. The levels of osteogenic differentiation-related proteins were analyzed using western blotting. Estrogen-deficient osteoporosis model mice were established, and treated with the two exosome preparations. Micro-computed tomography and hematoxylin and eosin staining were performed after 6 weeks. MicroRNAs in Exo1 and Exo2 were sequenced and analyzed using bioinformatic analyses. Compared with Exo1 group, Exo2 had a stronger osteogenic differentiation promoting effect, but a weaker proliferation promoting effect. In ovariectomy-induced osteoporosis mice, both Exo1 and Exo2 improved the tibial density and reversed osteoporosis in vivo. High-throughput microRNA sequencing identified 221 differentially expressed microRNAs in HucMSC-derived exosomes upon osteogenic induction as compared with the untreated control group. Importantly, we found that 41 of these microRNAs are potentially critical for MSC-secreted exosomes during osteogenic induction. Mechanistically, exosomal miRNAs derived from osteogenic induced-HucMSCs are involved in bone development and differentiation, such as osteoclast differentiation and the MAPK signaling pathway. The expression of hsa-mir-2110 and hsa-mir-328-3p gradually increased with prolonged osteogenic differentiation and regulated target genes associated with bone differentiation, suggesting that they are probably the most important osteogenesis regulatory microRNAs in exosomes. In conclusion, we examined the contribution of osteogenic induction to the function of exosomes secreted by HucMSCs following osteogenic differentiation in vitro and in vivo, and reveal the underlying molecular mechanisms of exosome action during osteoporosis.

scholarly journals miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A

2021 ◽  
Vol 9 ◽  
Author(s):  
Ning Wang ◽  
Xiao Han ◽  
Haoqing Yang ◽  
Dengsheng Xia ◽  
Zhipeng Fan
Keyword(s):  
Stem Cells ◽  
Dental Pulp ◽  
Binding Protein ◽  
Dental Pulp Stem Cells ◽  
Luciferase Reporter ◽  
Control Group ◽  
Alizarin Red ◽  
Odontogenic Differentiation ◽  
Human Dental Pulp ◽  
Tooth Tissue

Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation.Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A.Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs.Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3′UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

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