scholarly journals The miR-187 Induced Bone Reconstruction and Healing in a Mouse Model of Osteoporosis, and Accelerated Osteoblastic Differentiation of Human Multipotent Stromal Cells by Targeting BARX2

2020 ◽  
Author(s):  
Jun Zhang ◽  
Feng Pan ◽  
Benseng Tang ◽  
Jing Li ◽  
Zhengang Zha
Keyword(s):  
Mouse Model ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Osteoporotic Fractures ◽  
Fracture Repair ◽  
Luciferase Reporter ◽  
Blue Staining ◽  
Bone Reconstruction ◽  
Alizarin Red ◽  
Matrix Mineralization

Abstract Background: Multiple microRNAs (miRNAs) have been proven to regulate osteogenic differentiation by affecting the Runx2 signaling pathway. The intervention of miRNA can delay the progress of osteoporosis (OP) and induce fracture repair by affecting bone regeneration. However, the function and mechanism of miR-187 in osteoporotic fractures are still unknown. Methods: We first established the OP mouse model. Next, the BMD value was certified by iDXA. The miR-187 level in the OP mice and serum of OP patients was identified through qRT-PCR. Bone repair and bone healing were assessed through toluidine blue staining and X-ray, and BARX2 expression was also confirmed. Osteogenesis-related proteins, ALP activity, and the matrix mineralization state were evaluated by western blot, ALP staining, and Alizarin Red staining in hMSCs after transfection with miR-187 mimics, miR-187 inhibitor, or human BarH-like homeobox 2 (BARX2) siRNA. Moreover, the interplay between miR-187 and BARX2 was identified through the dual-luciferase reporter.Results: The BMD value was notably reduced in the OP mice, and miR-187 was markedly downregulated in the OP mice and serum of OP patients. Meanwhile, we proved that miR-187 induced bone reconstruction and healing, and downregulated BARX2 in the OP mouse model. We also proved that BARX2 was a direct target of miR-187, and could be significantly downregulated by miR-187. Furthermore, miR-187 induced osteogenic differentiation of hMSCs by targeting BARX2.Conclusions: The miR-187 might have a significant therapeutic effect in osteoporotic fractures. miR-187 accelerated osteogenic differentiation of hMSCs by directly regulating BARX2.

scholarly journals Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Xudong Wang ◽  
Taiqiu Chen ◽  
Zhihuai Deng ◽  
Wenjie Gao ◽  
Tongzhou Liang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Specific Gene ◽  
Biological Processes ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Dual Luciferase Reporter Assay

Abstract Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

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 Concentrated Growth Factors (CGF) Induce Osteogenic Differentiation in Human Bone Marrow Stem Cells

Biology ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 370
Author(s):  
Alessio Rochira ◽  
Luisa Siculella ◽  
Fabrizio Damiano ◽  
Andrea Palermo ◽  
Franco Ferrante ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Bone Marrow Stem Cells ◽  
Protein Quantification ◽  
Human Bone ◽  
Human Bone Marrow ◽  
Osteogenic Medium ◽  
Alizarin Red ◽  
Matrix Mineralization

Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.

scholarly journals MicroRNA-346-5p Regulates Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells by Inhibiting Transmembrane Protein 9

2020 ◽  
Vol 2020 ◽  
pp. 1-7
Author(s):  
Yicai Zhang ◽  
Yi Sun ◽  
Jinlong Liu ◽  
Yu Han ◽  
Jinglong Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Protein Level ◽  
Molecular Mechanisms ◽  
Transmembrane Protein ◽  
Luciferase Reporter ◽  
Alizarin Red ◽  
Protein Levels

The molecular mechanisms how bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into osteoblast need to be investigated. MicroRNAs (miRNAs) contribute to the osteogenic differentiation of BMSCs. However, the effect of miR-346-5p on osteogenic differentiation of BMSCs is not clear. This study is aimed at elucidating the underlying mechanism by which miR-346-5p regulates osteogenic differentiation of human BMSCs. Results of alkaline phosphatase (ALP) and Alizarin Red S (ARS) staining indicated that upregulation of miR-346-5p suppressed osteogenic differentiation of BMSCs, whereas downregulation of miR-346-5p enhanced this process. The protein levels of the osteoblastic markers Osterix and Runt-related transcription factor 2 (Runx2) were decreased in cells treated with miR-346-5p mimic at day 7 and day 14 after being differentiated. By contrast, downregulation of miR-346-5p elevated the protein levels of Osterix and Runx2. Moreover, a dual-luciferase reporter assay revealed that Transmembrane Protein 9 (TMEM9) was a target of miR-346-5p. In addition, the Western Blot results demonstrated that the TMEM9 protein level was significantly reduced by the miR-346-5p mimic whereas downregulation of miR-346-5p improved the protein level of TMEM9. These results together demonstrated that miR-346-5p served a key role in BMSC osteogenic differentiation of through targeting TMEM9, which may provide a novel target for clinical treatments of bone injury.

Adenovirus-mediated expression of vascular endothelial growth factor-a potentiates bone morphogenetic protein9-induced osteogenic differentiation and bone formation

2016 ◽  
Vol 397 (8) ◽  
pp. 765-775 ◽  
Author(s):  
Chang-jun Pi ◽  
Kai-lu Liang ◽  
Zhen-yong Ke ◽  
Fu Chen ◽  
Yun Cheng ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Bone Formation ◽  
Osteogenic Differentiation ◽  
Bone Repair ◽  
Osteoporotic Fractures ◽  
Angiogenic Factor ◽  
Vascular Endothelial ◽  
Akt Inhibitor ◽  
Endothelial Growth Factor

Abstract Mesenchymal stem cells (MSCs) are suitable seed cells for bone tissue engineering because they can self-renew and undergo differentiation into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Vascular endothelial growth factor-a (VEGF-a), an angiogenic factor, is also involved in osteogenesis and bone repair. However, the effects of VEGF-a on osteogenic MSCs differentiation remain unknown. It was previously reported that bone morphogenetic protein9 (BMP9) is one of the most important osteogenic BMPs. Here, we investigated the effects of VEGF-a on BMP9-induced osteogenesis with mouse embryo fibroblasts (MEFs). We found that endogenous VEGF-a expression was undetectable in MSCs. Adenovirus-mediated expression of VEGF-a in MEFs potentiated BMP9-induced early and late osteogenic markers, including alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In stem cell implantation assays, VEGF-a augmented BMP9-induced ectopic bone formation. VEGF-a in combination with BMP9 effectively increased the bone volume and osteogenic activity. However, the synergistic effect was efficiently abolished by the phosphoinositide 3-kinase (PI3K)/AKT inhibitor LY294002. These results demonstrated that BMP9 may crosstalk with VEGF-a through the PI3K/AKT signaling pathway to induce osteogenic differentiation in MEFs. Thus, our findings demonstrate the effects of VEGF-a on BMP9-induced bone formation and provide a new potential strategy for treating nonunion fractures, large segmental bony defects, and/or osteoporotic fractures.

scholarly journals Melatonin restores osteoporosis-impaired osteogenic potential of bone marrow mesenchymal stem cells and alleviates bone loss through the HGF/PTEN/Wnt/β-catenin axis

2021 ◽  
Vol 12 ◽  
pp. 204062232199568
Author(s):  
Jun Zhang ◽  
Guoliang Jia ◽  
Pan Xue ◽  
Zhengwei Li
Keyword(s):  
Bone Marrow ◽  
Bone Loss ◽  
Osteogenic Differentiation ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Osteogenic Potential ◽  
Enzyme Linked Immunosorbent Assay ◽  
Tartrate Resistant Acid Phosphatase ◽  
Micro Computed Tomography ◽  
Alizarin Red

Background: Previous studies reported that melatonin exerts its effect on mesenchymal stem cell (MSC) survival and differentiation into osteogenic and adipogenic lineage. In the current study we aimed to explore the effect of melatonin on osteoporosis and relevant mechanisms. Methods: Real-time qualitative polymerase chain reaction (RT-qPCR) and Western blot analysis were conducted to determine expression of HGF, PTEN, and osteoblast differentiation-related genes in ovariectomy (OVX)-induced osteoporosis mice and the isolated bone marrow MSCs (BMSCs). Pre-conditioning with melatonin (1 μmol/l, 10 μmol/l and 100 μmol/l) was carried out in OVX mice BMSCs. Bone microstructure was analyzed using micro-computed tomography and the contents of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase 5b (TRAP5b) were detected by enzyme-linked immunosorbent assay in serum. BMSC proliferation was measured by cell-counting kit (CCK)-8 assay. Alizarin red S (ARS) staining and ALP activity assay were performed to assess BMSC mineralization and calcification. The activity of the Wnt/β-catenin pathway was evaluated by dual-luciferase reporter assay. Results: Melatonin prevented bone loss in OVX mice. Melatonin increased ALP expression and reduced TRAP5b expression. HGF and β-catenin were downregulated, while PTEN was upregulated in the femur of OVX mice. Melatonin elevated HGF expression and then stimulated BMSC proliferation and osteogenic differentiation. Additionally, HGF diminished the expression of PTEN, resulting in activated Wnt/β-catenin pathway both in vitro and in vivo. Furthermore, melatonin was shown to ameliorate osteoporosis in OVX mice via the HGF/ PTEN/ Wnt/β-catenin axis. Conclusion: Melatonin could potentially enhance osteogenic differentiation of BMSCs and retard bone loss through the HGF/ PTEN/ Wnt/β-catenin axis.

scholarly journals SIRT6 Promotes Osteogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells Through Antagonizing DNMT1

2021 ◽  
Vol 9 ◽  
Author(s):  
Bo Jia ◽  
Jun Chen ◽  
Qin Wang ◽  
Xiang Sun ◽  
Jiusong Han ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Stem Cells ◽  
Notch Signaling ◽  
Osteogenic Differentiation ◽  
Dna Methyltransferases ◽  
Luciferase Reporter ◽  
Calcium Deposition ◽  
Alizarin Red

BackgroundAdipose-derived stem cells (ADSCs) are increasingly used in regenerative medicine because of their potential to differentiate into multiple cell types, including osteogenic lineages. Sirtuin protein 6 (SIRT6) is a nicotinamide adenine dinucleotide (NAD)-dependent deacetylase that plays important roles in cell differentiation. NOTCH signaling has also been reported to involve in osteogenic differentiation. However, the function of SIRT6 in osteogenic differentiation of ADSCs and its relation to the NOTCH signaling pathways are yet to be explored.MethodsThe in vitro study with human ADSCs (hADSCs) and in vivo experiments with nude mice have been performed. Alkaline phosphatase (ALP) assays and ALP staining were used to detect osteogenic activity. Alizarin Red staining was performed to detect calcium deposition induced by osteogenic differentiation of ADSCs. Western blot, RT-qPCR, luciferase reporter assay, and co-immunoprecipitation assay were applied to explore the relationship between of SIRT6, DNA methyltransferases (DNMTs) and NOTCHs.ResultsSIRT6 promoted ALP activity, enhanced mineralization and upregulated expression of osteogenic-related genes of hADSCs in vitro and in vivo. Further mechanistic studies showed that SIRT6 deacetylated DNMT1, leading to its unstability at protein level. The decreased expression of DNMT1 prevented the abnormal DNA methylation of NOTCH1 and NOTCH2, resulting in the upregulation of their transcription. SIRT6 overexpression partially suppressed the abnormal DNA methylation of NOTCH1 and NOTCH2 by antagonizing DNMT1, leading to an increased capacity of ADSCs for their osteogenic differentiation.ConclusionThis study demonstrates that SIRT6 physical interacts with the DNMT1 protein, deacetylating and destabilizing DNMT1 protein, leading to the activation of NOTCH1 and NOTCH2, Which in turn promotes the osteogenic differentiation of ADSCs.

scholarly journals MicroRNA-409-3p promotes osteoblastic differentiation via activation of Wnt/β-catenin signaling pathway by targeting SCAI

2020 ◽  
Author(s):  
Nan Chen ◽  
Hao Yang ◽  
Lijun Song ◽  
Hua Li ◽  
Yi Liu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Complementary Sequence ◽  
Alizarin Red ◽  
Gene Assay

Osteogenic differentiation is an important process of new bone formation, miR-409-3p has been reported to be upregulated in osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). To investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism, the expression of miR-409-3p in osteoblast (HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to SCAI in MSC-B was investigated by dual-luciferase reporter gene assay. MSC-B were selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase activity, alizarin red staining, and the expression of osteogenic markers in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. The Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs, showing an increasing time-dependent trend on the 0 and 21th day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3′UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI upregulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes. Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by downregulating SCAI expression.

scholarly journals MicroRNA Hsa-Let-7b Regulates the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting CTHRC1

2021 ◽  
Vol 2021 ◽  
pp. 1-15
Author(s):  
Lin Fu ◽  
Na Li ◽  
Yu Ye ◽  
Xiaying Ye ◽  
Tong Xiao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Osteogenic Differentiation ◽  
Periodontal Ligament ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Periodontal Ligament Stem Cells ◽  
Alizarin Red ◽  
Proliferation Ability ◽  
Polymerase Chain ◽  
Dual Luciferase Reporter Assay

Let-7 miRNA family has been proved as a key regulator of mesenchymal stem cells’ (MSCs’) biological features. However, whether let-7b could affect the differentiation or proliferation of periodontal ligament stem cells (PDLSCs) is still unknown. Here, we found that the expression of hsa-let-7b was visibly downregulated after mineralization induction of PDLSCs. After transfected with hsa-let-7b mimics or inhibitor reagent, the proliferation ability of PDLSCs was detected by cell counting kit-8 (CCK-8), flow cytometry, and 5-ethynyl-2-deoxyuridine (EdU) assay. On the other hand, the osteogenic differentiation capacity was detected by alkaline phosphatase (ALP) staining and activity, alizarin red staining, Western blot, and quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). We verified that hsa-let-7b did not significantly impact the proliferation ability of PDLSCs, but it could curb the osteogenic differentiation of PDLSCs. Besides, we predicted CTHRC1 acts as the downstream gene of hsa-let-7b to affect this process. Moreover, the combination of CTHRC1 and hsa-let-7b was verified by dual luciferase reporter assay. Our results demonstrated that the osteogenic differentiation of PDLSCs was enhanced after inhibiting hsa-let-7b, while was weakened after cotransfection with Si-CTHRC1. Collectively, hsa-let-7b can repress the osteogenic differentiation of PDLSCs by regulating CTHRC1.

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