scholarly journals FOXO3 is targeted by miR-223-3p and promotes osteogenic differentiation of bone marrow mesenchymal stem cells by enhancing autophagy

Human Cell ◽  
2020 ◽  
Vol 34 (1) ◽  
pp. 14-27
Author(s):  
Cheng Long ◽  
Shiqiang Cen ◽  
Zhou Zhong ◽  
Chang Zhou ◽  
Gang Zhong
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Dependent Manner ◽  
Bone Marrow Mscs ◽  
Related Factors ◽  
Alizarin Red ◽  
Forkhead Box ◽  
Marrow Mesenchymal Stem Cells

AbstractMesenchymal stem cells (MSCs) are a promising regenerative medicine. The roles of miRNAs in osteogenic differentiation of bone marrow MSCs (BM-MSCs) remained less reported. Forkhead Box O3 (FOXO3) and alkaline phosphatase (ALP) levels in the BM-MSCs were measured on 3, 7, and 14 days after osteogenic differentiation. After transfection of FOXO3 overexpression plasmids or siFOXO3 into BM-MSCs, factors related to osteogenic differentiation or cell autophagy were determined. Besides, 3-methyladenine or rapamycin, as well as miR-223-3p mimic or inhibitor were applied to further determine the effect of FOXO3 in BM-MSCs. FOXO3 and ALP levels were increased in a time-dependent manner with osteogenic differentiation, supported by Alizarin Red Staining. Furthermore, up-regulated FOXO3 increased levels of ALP and factors related to osteogenic differentiation by increasing levels of autophagy-related factors. FOXO3, targeted by miR-223-3p, reversed the effects of miR-223-3p on factors related to BM-MSC autophagy and osteogenic differentiation. Down-regulated miR-223-3p expression promoted osteogenic differentiation of BM-MSCs by enhancing autophagy via targeting FOXO3, suggesting the potential of miR-223-3p as a therapeutic target for enhancing bone functions.

scholarly journals Thrombin-activated platelet rich plasma (PRP) enhanced osteogenic differentiation of bone marrow mesenchymal stem cells by activing autophagy through miR-140-3p/SPRED2 axis

2020 ◽  
Author(s):  
Shuting Jiang ◽  
Hongyan Liu ◽  
Weiyan Zhu ◽  
Hui Yan ◽  
Beizhan Yan
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Abstract Background Mesenchymal stem cells transplantation gradually become a potential treatment for bone defect in clinic practice. This study aimed to investigate the molecular mechanism of PRP and autophagy for osteogenic differentiation in bone marrow mesenchymal stem cells (BMSCs). Methods Thrombin activated PRP was prepared and the BMSCs were treated with activated PRP with different concentration and transfected with miR-140-3p vector (mimics or inhibitor), si-SPRED2 or co-transfected with miR-140-3p inhibitor and si-SPRED2, respectively. qRT-PCR and Western blotting were used to determine the mRNA expression and protein expression. A luciferase reporter assay was conducted to identified the targeting relationship between iR-140-3p and SPRED2 Subsequently, cell proliferation was detected by MTT and ALP activity was also determined. Alizarin red staining was used for the evaluating the formation of calcium nodules. Results MiR-140-3p expression was found to be inhibited by PRP in a dose-dependent manner, besides, cell proliferation, ALP activity, the expression of COL-I, OPN, Runx2 and OCN, and the formation of calcium nodules related to osteogenic differentiation were enhanced by PRP. Subsequently, we found that PRP activated autophagy and up-regulated SPRED2 expression in BMSCs through suppressing miR-140-3p expression. Moreover, we confirmed that miR-140-3p targeted SPRED2 and negatively regulation its expression. Finally, the findings showed that inhibition of miR-140-3p enhanced cell proliferation, osteogenic differentiation and autophagy of BMSCs by negatively regulating SPRED2 expression. Conclusion Thrombin activated PRP accelerated osteogenic differentiation of BMSCs by activing autophagy through miR-140-3p/SPRED2 axis.

miRNA-126 Inhibits Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells (BMSCs)

2022 ◽  
Vol 12 (4) ◽  
pp. 794-799
Author(s):  
Le Chang ◽  
Wei Duan ◽  
Chuang Wang ◽  
Jian Zhang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Mrna Level ◽  
Alizarin Red ◽  
Runx2 Expression ◽  
Alp Activity ◽  
Smad4 Protein ◽  
Marrow Mesenchymal Stem Cells

This study was to determine whether microRNA (miRNA)-126 regulates osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). Rat BMSCs were extracted and stimulated for osteogenic differentiation. Functional experiments were conducted to assess miR-126’s impact on BMSCs differentiation. Western blot and RT-qPCR determined miR-126 expression. ALP activity detection and alizarin red staining detection were also performed. After osteogenic differentiation of BMSCs, miR-126 expression was gradually decreased over time. Overexpression of miR-26 decreased ALP activity, Notch signaling activity as well as declined Runx2 expression and calcium Salt nodules after treatment. Importantly, we found that Smad4 serves as a target of miR-126 while upregulation of the miRNA was accompanied with the decreased Smad4 protein expression without affecting the Smad4 mRNA level. In conclusion, miR-126 restrains osteogenic differentiation through inhibition of SMAD4 signaling, providing a novel insight into the mechanism.

Differential Expression of MicroRNA-3148 in Patients with Osteoporosis and Its Impacts on the Osteogenic Differentiation of Rat Bone Marrow Mesenchymal Stem Cells

2021 ◽  
Vol 11 (5) ◽  
pp. 957-962
Author(s):  
Ainiwaerjiang Damaola ◽  
Maerdan Aierken ◽  
Mieralimu Muertizha ◽  
Abudouaini Abudoureheman ◽  
Haishan Lin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Serum Samples ◽  
Alizarin Red ◽  
Differentiation Potential ◽  
Qrt Pcr ◽  
Marrow Mesenchymal Stem Cells ◽  
Rat Bone

We aimed to explore the effects of rat bone marrow mesenchymal stem cells (BMSCs) on osteogenic differentiation via analyzing miR-3148 expression in patients with osteoporosis. Realtime quantitative PCR was conducted for assessing microRNA-3148 expression. BMSCs from SD rats were transfected with microRNA-3148 mimics and microRNA-3148 inhibitor via liposomal trans-fection method utilizing Lipo2000, followed by analysis of microRNA-3148 level. After 10-days of osteogenic differentiation induction, alkaline phosphatase (ALP) staining and alizarin red (ARS) staining were done to investigate the osteogenic differentiation potential. Simultaneously, qRT-PCR measured the expression of osteogenesis marker genes (BMP and Runx2) in each group. qRT-PCR analysis revealed a high expression of miR-3148 in the bone tissue and the serum samples from patients with osteoporosis in comparison with healthy individuals. In addition, miRNA-3148 mimics could retard the osteogenic differentiation of BMSCs, while microRNA-3148 inhibitor could prompt the procedure. MicroRNA-3148 was highly expressed in the skeletal tissues and the serum samples from patients with osteoporosis and it could restrain the differentiation of BMSCs into osteoblasts, suggesting that it might be a novel therapeutic target for treating osteoporosis.

scholarly journals Polypeptides IGF-1C and P24 synergistically promote osteogenic differentiation of bone marrow mesenchymal stem cells in vitro through the p38 and JNK signaling pathways

2021 ◽  
Author(s):  
Gaoying Ran ◽  
Wei Fang ◽  
Lifang Zhang ◽  
Yuting Peng ◽  
Jiatong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bone Morphogenetic Protein 2 ◽  
Cell Counting ◽  
Signal Pathways ◽  
Alizarin Red ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Objectives: Insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein 2 (BMP-2) both promote osteogenesis of bone marrow mesenchymal stem cells (BMSCs). IGF-1C, the C domain peptide of IGF-1, and P24, a BMP-2-derived peptide, both have similar biological activities as their parent growth factors. This study aimed to investigate the effects and their mechanisms of polypeptides IGF-1C and P24 on the osteogenic differentiation of BMSCs. Methods: The optimum concentrations of IGF-IC and P24 were explored. The effects of the two polypeptides on the proliferation and osteogenic differentiation of BMSCs were examined using the Cell Counting Kit-8 (CCK-8), Alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red S staining, qPCR, and western blotting. In addition, specific pathway inhibitors were utilized to explore whether p38 and JNK pathways were involved in this process. Results: The optimal concentrations of action were both 50 g/ml. IGF-1C and P24 synergistically promoted the proliferation of BMSCs, increased ALP activity and the formation of calcified nodules and upregulated the mRNA and protein levels of osterix (Osx), runt-related transcription factor 2 (Runx2), and osteocalcin (Ocn), phosphorylation level of p38 and JNK proteins also improved. Inhibition of the pathways significantly reduced the activation of p38 and JNK, blocked the expression of Runx2 while inhibiting ALP activity and the formation of calcified nodules. Conclusions: These findings suggest IGF-1C and P24 synergistically promote the osteogenesis of BMSCs through activation of p38 and JNK signal pathways.

scholarly journals Osteogenic Differentiation of Pre-Conditioned Bone Marrow Mesenchymal Stem Cells with Nisin on the Modified Poly-L-Lactic-Acid Nanofibers

2021 ◽  
Author(s):  
Fariba Sadraei ◽  
Marzieh Ghollasi ◽  
Fatemeh Khakpai ◽  
Raheleh Halabian
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Lactic Acid ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Human bone marrow-derived mesenchymal stem (MSCs) cells are undifferentiated cells with the self-renewing ability and multi-lineage differentiation beneficial for regenerative medicine. Nano scaffolds are novel materials employed in bone repair and regeneration. Nisin is a prebiotic that can increase stem cells’ life span and proliferation. This study attempted to provide a proper strategy for bone marrow mesenchymal stem cells differentiation into the Osteocytes on a Poly‐L‐lactic‐acid scaffold (PLLA) after pretreating with probiotic Nisin. Methods: MSC osteogenic differentiation was evaluated by measuring Calcium, Alkaline phosphatase, and quantitative tests such as Real-Time PCR, Acridine Orange, Alizarin Red, Von Kossa, and others. Results: The result of the MTT test showed that the optimal dose of Nisin probiotic for the MSCs’ preconditioning was 200 IU/mL on the 1st, 3rd, and 5th days of culture. Real-time PCR data indicated that the expression rate of ALP, Osteonectin, Osteocalcin, and Collagen I have increased in the presence of Nisin, while the RUNX-2 gene expression has decreased. Furthermore, the results of Alizarin Red and Von Kossa tests, as well as Scanning electron microscopy (SEM), revealed that the cell proliferation in the preconditioned samples with Nisin increased significantly. Conclusions: The study concluded that the cell proliferation and differentiation increased in samples pretreated with Nisin on the PLLA Nano scaffolds.

scholarly journals Long non-coding RNA taurine upregulated gene 1 is downregulated in osteoporosis and influences the osteogenic differentiation of bone marrow mesenchymal stem cells

PeerJ ◽  
2021 ◽  
Vol 9 ◽  
pp. e11251
Author(s):  
Zhaowei Teng ◽  
Yun Zhu ◽  
Qinggang Hao ◽  
Xiaochao Yu ◽  
Yirong Teng ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Non Coding Rna ◽  
Marrow Mesenchymal Stem Cells ◽  
Long Non Coding Rna ◽  
Taurine Upregulated Gene 1

Background With aging, an imbalance in bone remodeling leading to increased bone resorption and decreased bone formation is thought to contribute to osteoporosis. Osteoblastic differentiation of bone marrow mesenchymal stem cells (BMMSCs) plays a vital role in the pathogenesis of osteoporosis. However, the detailed molecular mechanisms of osteoporosis remain incompletely understood. Given that long non-coding RNA taurine upregulated gene 1 (lnc TUG1) plays a critical role in the osteogenic differentiation, and microRNA-23b (miR-23b) as a putative sponge for lnc TUG1 has upregulated expression in osteoporosis. Therefore, this study investigated the roles of TUG1/miR-23b in osteoporotic pathology. Material and Methods TUG1 and miR-23b expression in the plasma of osteoporotic patients were evaluated by quantitative real-time PCR (qRT-PCR). The osteogenic differentiation in human BMMSCs was evaluated by qRT-PCR, western blot, Alizarin red staining after knockdown of TUG1 by small interfering RNA (siRNA) treatment. Results Decreased expression of TUG1 and increased expression of miR-23b evident in the plasma of patients with osteoporosis than in that of age- and sex-matched healthy controls. Additionally, increased miR-23b expression inhibited runt-related transcription factor 2 (RUNX2), osteocalcin, and osteopontin expression and reduced calcified nodule formation based on the results of qRT-PCR, western blot, and Alizarin Red S staining. Conclusion The study for the first time reported that silence of lncRNA TUG1 significantly suppressed the osteogenic differentiation of BMMSCs possibly by targeting the miR-23b/RUNX2 signaling pathway. This mechanism of TUG1/miR-23b/RUNX2 signaling within the osteogenic differentiation of BMMSCs might provide new insight for the development of lncRNA-directed diagnostic and therapeutic strategies for osteoporosis.

scholarly journals Morusin induces osteogenic differentiation of bone marrow mesenchymal stem cells by canonical Wnt/β-catenin pathway and prevents bone loss in an ovariectomized rat model

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ming Chen ◽  
Hui Han ◽  
Siqi Zhou ◽  
Yinxian Wen ◽  
Liaobin Chen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Nuclear Translocation ◽  
Osteoclast Differentiation ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background Osteoporosis (OP) is a metabolic bone disease due to the imbalance of osteogenesis and bone resorption, in which, bone marrow mesenchymal stem cells (BMSCs) have a significant effect as the seed cells. Recent research has shown the function of Morusin on inhibiting osteoclast differentiation in vitro. However, whether Morusin can regulate the osteogenic differentiation in addition to the proliferation of BMSCs remains unclear. Methods BMSCs were isolated from 4-week-old Wistar rats and then treated with different concentrations of Morusin for 3, 5, 7, and 14 days. The proliferation of BMSCs was detected by MTT assay. The effect of Morusin on osteogenic differentiation of BMSCs was detected by RT-qPCR, Western blotting, ALP, and Alizarin Red staining. The effect of Morusin on Wnt/β-catenin signaling pathway was analyzed by RT-qPCR, Western blotting, and immunofluorescence. Finally, in the ovariectomy-induced osteoporosis model, the anti-osteoporosis activity of Morusin was determined by micro-CT, HE, and immunohistochemistry. Results The results showed the function of 2.5–10 μM Morusin in the promotion of the proliferation in addition to osteogenic differentiation of BMSCs. Moreover, it also has an impact in activating the Wnt/β-catenin signaling pathway via inhibition of β-catenin phosphorylation as well as promotion of its nuclear translocation. Upon Dickkopf-related protein-1 (DKK-1, an inhibitor of the Wnt/β-catenin signaling pathway) was added to the Morusin, Morusin had a decreased stimulatory osteogenic effect on BMSCs. Finally, in the rat OP model, we found that Morusin could also exert anti-osteoporosis activity in vivo. Conclusions This study indicates the ability of Morusin in the promotion of osteogenic differentiation of BMSCs via the activation of Wnt/β-catenin signaling pathway and also shows the potential of Morusin to be an agent for osteoporosis treatment.

scholarly journals Effect of miR-192-5p on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Idiopathic Scoliosis by Regulating Wnt/β-catenin Signaling Pathway via RSPO1

2021 ◽  
Author(s):  
Yifan Yang ◽  
Jing Xu ◽  
Qingxin Su ◽  
Yiran Wu ◽  
Qizheng Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Alkaline Phosphatase ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Alizarin Red ◽  
Alkaline Phosphatase Staining ◽  
Marrow Mesenchymal Stem Cells ◽  
Related Proteins

Abstract BackgroundIdiopathic scoliosis (IS) is the most common structural scoliosis, which seriously affects not only patient’s physical and mental health but also quality of patient’s life. Abnormal osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is one of the causes of IS. However, the regulation mechanism of osteogenic differentiation of BMSCs in patients with IS remains to be further studied.MethodsSerum samples of 135 patients with IS were collected, and the expression of miRNA were detected by RT-qPCR. BMSCs from patients with IS were collected and the expression of miR-192-5p in BMSCs from IS patients and normal BMSCs was detected by RT-qPCR. Double luciferase reporter genes assay was used to verify the targeting relationship between miR-192-5p and RSPO1. The levels of RSPO1, osteogenic related proteins (OC, OPN and RUNX2) and Wnt/β-catenin signaling pathway related proteins (WNT3A and β-catenin) were detected by Western blotting. Alkaline phosphatase staining and alizarin red staining were used to evaluate the osteogenesis of BMSCs.ResultsmiR-192-5p was significantly up-regulated in serum and BMSCs of patients with IS. Alkaline phosphatase staining and alizarin red staining showed that miR-192-5p inhibitor promoted the osteogenic differentiation of BMSCs from IS patients. miR-192-5p targeted down-regulated the expression of RSPO1 in BMSCs from IS patients. In addition, overexpression of RSPO1 activated Wnt/β-catenin signaling pathway in BMSCs from IS patients. Furthermore, miR-192-5p/RSPO1 axis regulated levels of osteogenic related proteins (OC, OPN and RUNX2) in BMSCs from IS patients through Wnt/β-catenin signaling pathway, and affected the osteogenic differentiation of BMSCs.ConclusionmiR-192-5p, which was highly expressed in patients with IS, inhibited Wnt/β-catenin signaling pathway by down-regulating RSPO1 protein and then reduced the osteogenic differentiation ability of BMSCs.

scholarly journals CHNQD-00603 Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by the miR-452-3p-Mediated Autophagy Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Shanshan Xin ◽  
Shao-Ming Li ◽  
Ling Gao ◽  
Jing-Jing Zheng ◽  
Yan-Wei Wu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Bone Structure ◽  
Alizarin Red S ◽  
Hydroxyl Group ◽  
Alizarin Red ◽  
Qrt Pcr ◽  
Marrow Mesenchymal Stem Cells

Background: Periodontitis is a chronic and progressive disease accompanied by bone loss. It is still a challenge to restore the bone structure. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) plays a decisive role in bone restoration and regeneration. Marine natural products (MNPs) have multiple biological activities, including anti-tumor and anti-inflammatory properties. However, the exploration of MNPs in osteogenesis is far from sufficient.Methods: We obtained a series of derivatives through structural optimization from 4-phenyl-3,4-dihydroquinolin-2(1H)-one alkaloid isolated from Scopulariopsis sp. Some preliminary cytological experiments showed that CHNQD-00603, obtained by adding a methoxy group to the position C3 and a hydroxyl group to the position C4 of 4-phenyl-3,4-dihydroquinolin-2(1H)-one, might promote the osteogenic differentiation of BMSCs. To further investigate the effects of CHNQD-00603 on BMSCs, we performed a CCK-8 assay and qRT-PCR, alkaline phosphatase staining (ALP), and alizarin red S staining to assess the cytotoxicity and the ability of osteogenic differentiation of CHNQD-00603. The autophagy level was assessed and validated by WB, qRT-PCR, and transmission electron microscopy. Then, 3-methyladenine (3-MA) was added to further examine the role of autophagy. Based on the expression of autophagy-related genes, we predicted and examined the potential miRNAs by bioinformatics.Results: CCK-8 assay showed that CHNQD-00603 at 1 µg/ml did not influence BMSCs activity. However, the proliferation rate decreased from the seventh day. qRT-PCR, ALP staining, ALP activity assay, and Alizarin red S staining showed that the best concentration of CHNQD-00603 to promote osteogenic differentiation was 1 µg/ml. Further investigations indicated that CHNQD-00603 activated autophagy, and the inhibition of autophagy by 3-MA attenuated CHNQD-00603-enhanced osteogenic differentiation. Subsequently, the findings from bioinformatics and qRT-PCR indicated that miR-452-3p might be a regulator of autophagy and osteogenesis. Furthermore, we transfected BMSCs with miR-452-3p NC and mimics separately to further determine the function of miR-452-3p. The data showed that the overexpression of miR-452-3p moderated the level of autophagy and osteogenic differentiation of CHNQD-00603-treated BMSCs.Conclusion: Our data suggested that CHNQD-00603 promoted the osteogenic differentiation of BMSCs by enhancing autophagy. Meanwhile, miR-452-3p played a regulatory role in this process.

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