miR-150 Affects Wnt/β-Catenin Pathway Activity and Regulates MSC Osteogenic Differentiation

2019 ◽  
Vol 9 (10) ◽  
pp. 1339-1345 ◽  
Author(s):  
Junsheng Wang ◽  
Xiaowu Hu ◽  
Qing Ji ◽  
Bai Zheng ◽  
Lixin Huang
Keyword(s):  
Stem Cells ◽  
Osteoblast Differentiation ◽  
Bioinformatics Analysis ◽  
Reporter Assay ◽  
Luciferase Gene ◽  
Alizarin Red ◽  
Pathway Activity ◽  
Gene Reporter Assay ◽  
The Relationship ◽  
Luciferase Gene Reporter Assay

The Wnt/β-catenin pathway promotes osteoblast differentiation. Bioinformatics analysis showed a targeting relationship between miR-150 and β-catenin 3′-UTR. This study investigated miR-150's role in β-catenin expression and osteoblast differentiation of Adipose-Derived Mesenchymal Stem Cells (ADMSCs). Differentiation of ADMSCs into osteoblasts was induced and the expression of miR-150, β-catenin, RUNX2 and OPN on the 0th, 9th and 18th day during differentiation was detected. The dual luciferase gene reporter assay validated the relationship between miR-150 and β-catenin. ADMSCs were divided into angomir-NC group and angomir-150 group, and then induced for differentiation for 18 days followed by measuring the level of miR-150, β-catenin, RUNX2 and OPN and the degree of osteoblast differentiation by alizarin red staining. Compared with Day 0, miR-150 expression was gradually decreased on 9th and 18th day of osteoblast differentiation, and the expression of β-catenin, RUNX2 and OPN was gradually increased. There was a targeted regulation relationship between miR-150 and β-catenin. Compared with angomir-NC group, miR-150 expression was significantly increased in angomir-150 transfection group with decreased expression of β-catenin, RUNX2 and OPN protein and reduced degree of alizarin red staining. miR-150 expression is decreased and β-catenin expression is increased during osteoblast differentiation of ADMSCs. Up-regulation of miR-150 expression attenuates the ability of ADMSCs to differentiate into osteoblasts by inhibiting β-catenin expression and Wnt/β-catenin pathway.

scholarly journals Negative Regulation of Serine Threonine Kinase 11 (STK11) through miR-100 in Head and Neck Cancer

Genes ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 1058
Author(s):  
Gabriela Figueroa-González ◽  
José F. Carrillo-Hernández ◽  
Itzel Perez-Rodriguez ◽  
David Cantú de León ◽  
Alma D. Campos-Parra ◽  
...  
Keyword(s):  
Head And Neck Cancer ◽  
Head And Neck ◽  
Neck Cancer ◽  
Reporter Assay ◽  
Luciferase Gene ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Gene Reporter Assay ◽  
Post Transcriptional Regulation ◽  
Luciferase Gene Reporter Assay

Background: Serine Threonine Kinase 11 (STK11), also known as LKB1, is a tumor suppressor gene that regulates several biological processes such as apoptosis, energetic metabolism, proliferation, invasion, and migration. During malignant progression, different types of cancer inhibit STK11 function by mutation or epigenetic inactivation. In Head and Neck Cancer, it is unclear what mechanism is involved in decreasing STK11 levels. Thus, the present work aims to determine whether STK11 expression might be regulated through epigenetic or post-translational mechanisms. Methods: Expression levels and methylation status for STK11 were analyzed in 59 cases of head and neck cancer and 10 healthy tissue counterparts. Afterward, we sought to identify candidate miRNAs exerting post-transcriptional regulation of STK11. Then, we assessed a luciferase gene reporter assay to know if miRNAs directly target STK11 mRNA. The expression levels of the clinical significance of mir-100-3p, -5p, and STK11 in 495 HNC specimens obtained from the TCGA database were further analyzed. Finally, the Kaplan–Meier method was used to estimate the prognostic significance of the miRNAs for Overall Survival, and survival curves were compared through the log-rank test. Results: STK11 was under-expressed, and its promoter region was demethylated or partially methylated. miR-17-5p, miR-106a-5p, miR-100-3p, and miR-100-5p could be negative regulators of STK11. Our experimental data suggested evidence that miR-100-3p and -5p were over-expressed in analyzed tumor patient samples. Luciferase gene reporter assay experiments showed that miR-100-3p targets and down-regulates STK11 mRNA directly. With respect to overall survival, STK11 expression level was significant for predicting clinical outcomes. Conclusion: This is, to our knowledge, the first report of miR-100-3p targeting STK11 in HNC. Together, these findings may support the importance of regulation of STK11 through post-transcriptional regulation in HNC and the possible contribution to the carcinogenesis process in this neoplasia.

scholarly journals MiR-214 Attenuates Osteogenic Differentiation of Mesenchymal Stem Cells via Targeting FGFR1

2016 ◽  
Vol 38 (2) ◽  
pp. 809-820 ◽  
Author(s):  
Lei Yang ◽  
Dawei Ge ◽  
Xiaojian Cao ◽  
Yingbin Ge ◽  
Hongtao Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Postmenopausal Osteoporosis ◽  
Osteoblast Differentiation ◽  
Luciferase Reporter Assay ◽  
Luciferase Reporter ◽  
Reporter Assay ◽  
Matrix Mineralization ◽  
Direct Target

Background/Aims: Postmenopausal osteoporosis is closely associated with reduction in the differentiation of mesenchymal stem cells (MSCs) into osteoblasts. Previous studies have demonstrated that miR-214 plays an important role in the genesis and development of postmenopausal osteoporosis. Here, we performed this study to investigate the potential mechanism by which miR-214 regulates osteoblast differentiation of MSCs. Methods: First, we explored the expression of miR-214 in MSCs of osteoporotic mice. Next, we examined the change of miR-214 during osteoblast differentiation of MSCs. Then, MSCs were infected with lentiviral vectors expressing miR-214 or miR-214 sponge to investigate the effect of miR-214 on osteoblast differentiation of MSCs. Further, bioinformatics analysis and luciferase reporter assay were performed to identify and validate the target gene of miR-214. Results: MiR-214 was up-regulated in MSCs of osteoporotic mice and down-regulated during osteoblast differentiation of MSCs. Furthermore, overexpression of miR-214 inhibited osteoblast differentiation of MSCs in vitro, whereas inhibition of miR-214 function promoted this process, evidenced by increased expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Bioinformatics, Western blot analysis and luciferase reporter assay demonstrated that FGFR1 is a direct target of miR-214. Conclusions: MiR-214 attenuates osteogenesis by inhibiting the FGFR1/FGF signaling pathway. Our findings suggest that targeting miR-214 promises to be a potential therapy in treatment of postmenopausal osteoporosis.

Exosomes from bone marrow mesenchymal stem cells promoted osteogenic differentiation by delivering miR-196a that targeted Dkk1 to activate Wnt/β-catenin pathway

2020 ◽  
Author(s):  
Zhi Peng ◽  
Zhenkai Lou ◽  
Zhongjie Li ◽  
Shaobo Li ◽  
Kaishun Yang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Negative Regulator ◽  
Luciferase Assay ◽  
Alizarin Red ◽  
Marrow Mesenchymal Stem Cells

Abstract Background: Osteoporosis is the most common bone metabolic disease. Emerging evidence suggests that exosomes are secreted by diverse cells such as bone marrow mesenchymal stem cells (BMSCs), and play important role in cell-to-cell communication and tissue homeostasis. Recently, the discovery of exosomes has attracted attention in the field of bone remodeling. Methods: The exosomes were extracted from BMSCs and labeled by PKH-67, and then incubated with hFOB1.19 cells to investigate the miR-196a function on the osteoblast differentiation of hFOB1.19. The osteoblast differentiation was detected via alizarin red staining and the expression of osteoblast genes were detected by western blot. The cell apoptosis was detected by flow cytometer. The target relationship of miR-196a and Dickkopf-1 (Dkk1) were verified by luciferase assay and western blot. Results: Here we demonstrated that exosomes extracted from BMSCs (BMSC-exo) significantly promoted hFOB1.19 differentiation to osteoblasts. We found that BMSC-exo were enriched with miR-196a and delivered miR-196a to hFOB1.19 cells to inhibit its target Dkk1, which is a negative regulator of Wnt/β-catenin pathway. Conclusion: BMSC-exo activated Wnt/β-catenin pathway to promote osteogenic differentiation, while BMSC-exo failed to exert the effects when miR-196a was deprived. In conclusion, miR-196a delivered by exosomes from BMSCs plays an essential role in enhancing osteoblastic differentiation by targeting Dkk1 to activate Wnt/β-catenin pathway.

scholarly journals Leonurine Promotes the Osteoblast Differentiation of Rat BMSCs by Activation of Autophagy via the PI3K/Akt/mTOR Pathway

2021 ◽  
Vol 9 ◽  
Author(s):  
Bingkun Zhao ◽  
Qian Peng ◽  
Enoch Hin Lok Poon ◽  
Fubo Chen ◽  
Rong Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Mtor Pathway ◽  
Control Group ◽  
Alizarin Red ◽  
Qrt Pcr

BackgroundLeonurine, a major bioactive component from Herba leonuri, has been shown to exhibit anti-inflammatory and antioxidant effects. The aim of this study was to investigate the effect of leonurine on bone marrow-derived mesenchymal stem cells (BMSCs) as a therapeutic approach for treating osteoporosis.Materials and MethodsRat bone marrow-derived mesenchymal stem cells (rBMSCs) were isolated from 4-weeks-old Sprague–Dawley rats. The cytocompatibility of leonurine on rBMSCs was tested via CCK-8 assays and flow cytometric analyses. The effects of leonurine on rBMSC osteogenic differentiation were analyzed via ALP staining, Alizarin red staining, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Additionally, autophagy-related markers were examined via qRT-PCR and Western blot analyses of rBMSCs during osteogenic differentiation with leonurine and with or without 3-methyladenine (3-MA) as an autophagic inhibitor. Finally, the PI3K/Akt/mTOR signaling pathway was evaluated during rBMSC osteogenesis.ResultsLeonurine at 2–100 μM promoted the proliferation of rBMSCs. ALP and Alizarin red staining results showed that 10 μM leonurine promoted rBMSC osteoblastic differentiation, which was consistent with the qRT-PCR and Western blot results. Compared with those of the control group, the mRNA and protein levels of Atg5, Atg7, and LC3 were upregulated in the rBMSCs upon leonurine treatment. Furthermore, leonurine rescued rBMSC autophagy after inhibition by 3-MA. Additionally, the PI3K/AKT/mTOR pathway was activated in rBMSCs upon leonurine treatment.ConclusionLeonurine promotes the osteoblast differentiation of rBMSCs by activating autophagy, which depends on the PI3K/Akt/mTOR pathway. Our results suggest that leonurine may be a potential treatment for osteoporosis.

miR-143 Inhibits Osteogenic Differentiation of Umbilical Cord Mesenchymal Stem Cell by Targeting Zinc Fingers and Homeoboxes 3

2019 ◽  
Vol 9 (11) ◽  
pp. 1550-1556
Author(s):  
Haiying Chen ◽  
Lei Zhang ◽  
Lijun Tao
Keyword(s):  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Umbilical Cord ◽  
Osteoblast Differentiation ◽  
Bioinformatics Analysis ◽  
Zinc Fingers ◽  
Alizarin Red ◽  
Msc Differentiation

The transcription regulating factor-zinc fingers and homeoboxes 3 (ZHX3) involve in osteoblast differentiation. miR-143 regulates osteoblast differentiation. Bioinformatics analysis revealed a relationship between miR-143 and the 3′ -UTR of ZHX3. This study assessed miR-143's role in UC-MSC differentiation. The UC-MSCs were induced to differentiate into osteoblasts. The expressions of miR-143, ZHX3, RUNX2, Osteopontin (OPN) and Osteocalcin (OC) genes were detected by qRTPCR on days 0, 20 and 30. The UC-MSCs cells were divided into agomir-NC group and miR-143 agomir group. After 30 days of differentiation, the expression of ZHX3, RUNX2, OPN and OC protein was detected by western blot. During differentiation of UC-MSCs into osteoblasts, miR-143 expression was decreased, ZHX3 expression, ALP activity and alizarin red staining was increased. There was a targeted regulation relationship between miR-143 and ZHX3. Transfection of agomir143 significantly downregulated ZHX3, RUNX2, OPN, OC genes and proteins, significantly reduced alizarin red staining, and decreased the ability of osteoblast differentiation. Decreased miR-143 expression plays a role in up-regulating ZHX3 expression and promoting osteoblast differentiation of UC-MSCs. Overexpression of miR-143 can inhibit UC-MSCs differentiation into osteoblasts by targeting ZHX3.

Valproic acid, A Potential Inducer of Osteogenesis in Mouse Mesenchymal Stem Cells

2020 ◽  
Vol 14 (1) ◽  
pp. 27-35 ◽  
Author(s):  
Narayanan Akshaya ◽  
Prakash Prasith ◽  
Balakrishnan Abinaya ◽  
Badrinath Ashwin ◽  
S.V. Chandran ◽  
...  
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Mesenchymal Stem Cells ◽  
Osteoblast Differentiation ◽  
Molecular Mechanisms ◽  
Cellular Level ◽  
Mitogen Activated Protein Kinase ◽  
Marker Genes ◽  
Herbaceous Plant ◽  
Alizarin Red

Background: Recent reports have unveiled the potential of flavonoids to enhance bone formation and assuage bone resorption due to their involvement in cell signaling pathways. They also act as an effective alternative to circumvent the disadvantages associated with existing treatment methods, which has increased their scope in orthopedic research. Valproic acid (VA, 2-propylpentanoic acid) is one such flavonoid, obtained from an herbaceous plant, used in the treatment of epilepsy and various types of seizures. Objective: In this study, the role of VA in osteogenesis and the molecular mechanisms underpinning its action in mouse mesenchymal stem cells (mMSCs) were determined. Methods & Results: Cytotoxic studies validated VA’s amiable nature in mMSCs. Alizarin red and von Kossa staining results showed an increased deposition of calcium phosphate in VA-treated mMSCs, which confirmed the occurrence of osteoblast differentiation and mineralization at a cellular level. At the molecular level, there were increased levels of expression of Runx2, a vital bone transcription factor, and other major osteoblast differentiation marker genes in the VAtreated mMSCs. Further, VA-treatment in mMSCs upregulated mir-21 and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway, which might be essential for the expression/activity of Runx2. Conclusion: Thus, the current study confirmed the osteoinductive nature of VA at the cellular and molecular levels, opening the possibility for its application in bone therapeutics with mir-21.

miR-218 Regulates Glycogen Synthase Kinase-3β and Promotes Differentiation of Human Bone Marrow Mesenchymal Stem Cells into Osteoblasts

2020 ◽  
Vol 10 (2) ◽  
pp. 176-181
Author(s):  
Long Ling ◽  
Hailan Hu ◽  
Ram Ishwar Yadav ◽  
Jianliang Gao ◽  
Xiao Wei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteoblast Differentiation ◽  
Glycogen Synthase ◽  
Induction Medium ◽  
Alizarin Red ◽  
Osteogenic Induction Medium ◽  
Marrow Mesenchymal Stem Cells ◽  
Osteogenic Induction

miR-218 is associated with osteogenesis. Bioinformatics analysis showed a targeting relationship between miR-218 and GSK-3β 3′-UTR. Our study assessed whether miR-218 regulates GSK-3β expression and affects osteoblast differentiation of bone marrow mesenchymal stem cells (BMSCs). Osteogenic induction medium was used to induce BMSCs to differentiate into osteoblasts. miR-218, GSK-3β, β-catenin and RUNX2 level was detected during D10 and D20 differentiation. BMSCs cells were divided into antagomir-NC and antagomir-218, and induced to differentiate for 20 days followed by analysis of GSK-3β, β-catenin and RUNX2 level, osteogenesis and cell differentiation by the alizarin red staining. Compared with pre-differentiation,the expression of miR-218, β-catenin and RUNX2 was gradually increased and GSK-3β expression was decreased during the differentiation of BMSCs into osteoblasts. There was a targeted regulation relationship between miR-218 and GSK-3β. Compared with the antagomir-NC group, GSK-3β protein expression was increased in antagomir-218 transfection group, with decreased the expression of β-catenin and RUNX2 protein, reduced ALP activity as well as weakened staining degree of alizarin red. GSK-3β expression is decreased and miR-218 expression is increased during osteoblast differentiation of BMSCs. Inhibition of miR-218 expression can up-regulate GSK-3β expression and attenuate the ability of BMSCs to differentiate into osteoblasts.

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.

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