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 MiR-27a-3p promotes the osteogenic differentiation by activating CRY2/ERK1/2 axis

2021 ◽  
Vol 27 (1) ◽  
Author(s):  
Li-Rong Ren ◽  
Ru-Bin Yao ◽  
Shi-Yong Wang ◽  
Xiang-Dong Gong ◽  
Ji-Tao Xu ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Inhibitory Effect ◽  
Luciferase Reporter ◽  
Induction Medium ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Protein Levels ◽  
Phosphorylation Level ◽  
Gene Assay

Abstract Background Osteoporosis seriously disturbs the life of people. Meanwhile, inhibition or weakening of osteogenic differentiation is one of the important factors in the pathogenesis of osteoporosis. It was reported that miR-27a-3p reduced the symptoms of osteoporosis. However, the mechanism by which miR-27a-3p in osteogenic differentiation remains largely unknown. Methods To induce the osteogenic differentiation in MC3T3-E1 cells, cells were treated with osteogenic induction medium (OIM). RT-qPCR was used to evaluate the mRNA expression of miR-27a-3p and CRY2 in cells. The protein levels of CRY2, Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin (OCN) and the phosphorylation level of extracellular regulated protein kinases (ERK) 1/2 in MC3T3-E1 cells were evaluated by western blotting. Meanwhile, calcium nodules and ALP activity were tested by alizarin red staining and ALP kit, respectively. Luciferase reporter gene assay was used to analyze the correlation between CRY2 and miR-27a-3p. Results The expression of miR-27a-3p and the phosphorylation level of ERK1/2 were increased by OIM in MC3T3-E1 cells, while CRY2 expression was decreased. In addition, OIM-induced increase of calcified nodules, ALP content and osteogenesis-related protein expression was significantly reversed by downregulation of miR-27a-3p and overexpression of CRY2. In addition, miR-27a-3p directly targeted CRY2 and negatively regulated CRY2. Meanwhile, the inhibitory effect of miR-27a-3p inhibitor on osteogenic differentiation was reversed by knockdown of CRY2 or using honokiol (ERK1/2 signal activator). Furthermore, miR-27a-3p significantly inhibited the apoptosis of MC3T3-E1 cells treated by OIM. Taken together, miR-27a-3p/CRY2/ERK axis plays an important role in osteoblast differentiation. Conclusions MiR-27a-3p promoted osteoblast differentiation via mediation of CRY2/ERK1/2 axis. Thereby, miR-27a-3p might serve as a new target for the treatment of osteoporosis.

scholarly journals Plastrum Testudinis Extracts Promote BMSC Proliferation and Osteogenic Differentiation by Regulating Let-7f-5p and the TNFR2/PI3K/AKT Signaling Pathway

2018 ◽  
Vol 47 (6) ◽  
pp. 2307-2318 ◽  
Author(s):  
Geng-Yang Shen ◽  
Hui Ren ◽  
Jin-Jing Huang ◽  
Zhi-Da Zhang ◽  
Wen-Hua Zhao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Akt Signaling ◽  
Cell Counting ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Alizarin Red

Background/Aims: Plastrum testudinis extracts (PTE) show osteoprotective effects on glucocorticoid-induced osteoporosis in vivo and in vitro. However, the underlying molecular mechanism of PTE in promoting osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is unclear. Methods: BMSC proliferation was investigated using the Cell Counting Kit-8 assay. BMSC differentiation and osteogenic mineralization were assayed using alkaline phosphatase and Alizarin red staining, respectively. The mRNA expression levels of Let-7f-5p, Tnfr2, Traf2, Pi3k, Akt, β-catenin, Gsk3β, Runx2, and Ocn were measured using real time quantitative polymerase chain reaction. Protein levels of TNFR2, TRAF2, p-PI3K, p-AKT, p-β-CATENIN, and p-GSK3β were analyzed by western blotting. The functional relationship of Let-7f-5p and Tnfr2 was determined by luciferase reporter assays. Results: The optimum concentration for PTE was 30 μg/ml. PTE significantly promoted BMSC osteogenic differentiation and mineralization after 7 and 14 days in culture, respectively. The combination of PTE and osteogenic induction exhibited significant synergy. PTE upregulated Let-7f-5p, β-catenin, Runx2, and Ocn mRNA expression, and downregulated Tnfr2, Traf2, Pi3k, Akt, and Gsk3β mRNA expression. PTE inhibited TNFR2, TRAF2, and p-β-CATENIN protein expression, and promoted p-PI3K, p-AKT, and p-GSK3β protein expression. In addition, Tnfr2 was a functional target of Let-7f-5p in 293T cells. Conclusions: Our results suggested that PTE may promote BMSC proliferation and osteogenic differentiation via a mechanism associated with the regulation of Let-7f-5p and the TNFR2/PI3K/AKT signaling pathway.

MiR-486-3p promotes osteogenic differentiation of BMSCs by targeting CTNNBIP1 and activating Wnt/β-catenin pathway

2021 ◽  
Author(s):  
Zheng Zhang ◽  
Weiwei Jiang ◽  
Miao Hu ◽  
Yichen Meng ◽  
Jun Ma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Osteoblastic Differentiation ◽  
Luciferase Reporter ◽  
Negative Effects ◽  
Alizarin Red ◽  
Interacting Protein ◽  
Marrow Mesenchymal Stem Cells ◽  
Osteogenic Gene Expression ◽  
Canonical Wnt

Abstract Background Dysfunction in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) leads to bone loss/osteoporosis. CTNNBIP1 (Catenin beta interacting protein 1) is an inhibitor of Wnt/β-catenin signaling, whose role in osteogenesis remains elusive. This study aims to reveal the effects of miR-486-3p/CTNNBIP1 in osteogenesis. Methods Bone marrow samples from control and osteoporosis patients were collected and ovariectomy was performed on mice and levels of CTNNBIP1 and miR-486-3p levels were assessed. Dual-luciferase reporter assay was used to confirm their interactions. MiR-486-3p mimics/inhibitor or CTNNBIP1 overexpression lentivirus were transfected in human BMSCs (hBMSCs) and then osteogenic assay was performed. Alizarin red S (ARS) and Alkaline phosphatase (ALP) intensity with osteogenic gene expression including Runx2, Alp, Bglap and OCN was measured. Key proteins in Wnt/β-catenin pathway including active β-catenin, Bcl-2 and Cyclin D1 were assessed. Results CTNNBIP1, an inhibitor of Wnt/β-catenin signaling was upregulated while miR-486-3p was downregulated in ovariectomized (OVX) mice. CTNNBIP1 was confirmed as a target of miR-486-3p. MiR-486-3p overexpression promoted but miR-486-3p knockdown suppressed osteogenic differentiation and Wnt/β-catenin pathway. Rescue experiments further elucidated that the negative effects of CTNNBIP1 overexpression on osteoblastic differentiation and canonical Wnt signaling could be reversed by miR-486-3p mimics. Conclusion This study demonstrated that miR-486-3p sponges CTNNBIP1 thus activating Wnt/β-catenin signaling pathway to promote osteogenesis of BMSCs.

scholarly journals miR-129-5p Promotes Osteogenic Differentiation of BMSCs and Bone Regeneration via Repressing Dkk3

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Changming Zhao ◽  
Yulin Gu ◽  
Yan Wang ◽  
Qiaozhen Qin ◽  
Ting Wang ◽  
...  
Keyword(s):  
Western Blot ◽  
Bone Regeneration ◽  
Osteogenic Differentiation ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alizarin Red ◽  
Gene Assay

Objective. Accumulating evidence indicates that microRNAs (miRNAs) play crucial roles in osteogenic differentiation. However, the associated mechanisms remain elusive. This paper is aimed at exploring the role of miR-129-5p in regulating bone marrow mesenchymal stem cell (BMSC) differentiation and bone regeneration in vivo and in vitro. Methods. BMSCs were transduced by miR-129-5p mimic, miR-129-5p inhibitor, and negative control lentivirus. The ability of BMSC differentiation to osteoblast was tested by alkaline phosphatase (ALP) and alizarin red staining (ARS). The expression of osteogenic genes (Runx2, Bmp2, and OCN) was examined via quantitative RT-PCR and western blot. A mouse model of calvaria defect was investigated by Micro-CT, immunohistochemistry, and histological examination. The luciferase reporter gene assay was performed to confirm the binding between Dkk3 and miR-129-5p. For the transfection experiments, lipofectamine 3000 was used to transfect pcDNA-Dkk3 into BMSCs to overexpress Dkk3. Coimmunoprecipitation and immunofluorescent localization assay were included for exploring the role of Dkk3 and β-catenin. Results. miR-129-5p was induced in BMSCs and MSC cell line C3H10T1/2 cells under osteogenic medium. Overexpression of miR-129-5p significantly promoted osteogenic differentiation of BMSCs in vitro. Moreover, BMSCs transduced with miR-129-5p mimic exhibited better bone regeneration compared with BMSCs transduced with control counterpart in vivo. Luciferase and western blot data showed that Dickkopf3 (Dkk3) is a target gene of miR-129-5p and the expression of Dkk3 was inhibited in BMSCs transduced with miR-129-5p mimic but enhanced in BMSCs transduced with miR-129-5p inhibitor. In addition, Dkk3 interacted with β-catenin directly. Conclusions. miR-129-5p promotes osteogenic differentiation of BMSCs and bone regeneration, and miR-129-5p/Dkk3 axis may be new potential targets for the treatment of bone defect and bone loss.

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.

Overexpressed miR-375-Loaded Restrains Development of Cervical Cancer Through Down-Regulation of Frizzled Class Receptor 4 (FZD4) with Liposome Nanoparticle as a Carrier

2021 ◽  
Vol 17 (9) ◽  
pp. 1882-1889
Author(s):  
Suqin Wang ◽  
Lina Xu ◽  
Zhiqiang Zhang ◽  
Ping Wang ◽  
Rong Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cervical Cancer ◽  
Cell Proliferation ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Gene Assay ◽  
M Phase ◽  
The Impact

Dysregulation expression of miR-375 is noted to correlate with progression of cervical cancer. This study attempted to investigate the impact of overexpressed miR-375-loaded liposome nanoparticles on proliferation of cervical cancer (CC), to provide an insight on pathogenesis of CC disorder. CC cells were co-cultured with pure liposome nanoparticles (empty vector group), miR-375 agonist-loaded liposome nanoparticles, or transfected with miR-375 antagonist. Besides, some cells were exposed to TGF-β/Smads signaling pathway inhibitor or activator whilst cell proliferation was assessed by MTT assay, and expressions of FZD4 and miR-375 were determined. Western blot analysis was carried out to detect the expression of TGF-β pathway factors (TGF-β, Smad2, Smad7, p-Smad2) and its downstream Smads pathway. The interaction between miR-375 and FZD4 was evaluated by dual-luciferase reporter gene assay. Overexpression of miR-375 induced arrest at the G0/G1 phase of cell cycle and elevation of Smad2 protein expression (P <0.05), with lower expressions of TGF-β, Smad7, p-Smad2, and FZD4, while transfection with miR-375 inhibitor exhibited opposite activity. Presence of miR-375 agonist-loaded liposome nanoparticles induced decreased cell proliferation. There was a targeting relationship between miR-375 and FZD4, and administration with TGF-β/Smads agonist resulted in increased miR-375 and Smad2 expressions, as well as decreased TGF-β, Smad7, p-Smad2, FZD4 protein expression, and the number of S phase and G2/M phase cells (P < 0.05). The signaling inhibitor oppositely suppressed cell proliferation decreasing miR-375 expression. miR-375-loaded liposome nanoparticles activated TGF-β/Smads signaling pathway to restrain cell cycle and suppress cell division, and proliferation through targeting FZD4 in CC. Its molecular mechanism is related to activation of TGF-β/Smads signaling pathway.

scholarly journals Bone Marrow Mesenchymal Stem Cell-Derived Exosomal miR-25 Regulates the Ubiquitination and Degradation of Runx2 by SMURF1 to Promote Fracture Healing in Mice

2020 ◽  
Vol 7 ◽  
Author(s):  
Yikun Jiang ◽  
Jun Zhang ◽  
Zhengwei Li ◽  
Guoliang Jia
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mesenchymal Stem Cell ◽  
Fracture Healing ◽  
Osteogenic Differentiation ◽  
Bioinformatics Analysis ◽  
Luciferase Reporter ◽  
Gene Assay ◽  
And Migration ◽  
Proliferation And Migration

Recent evidence has demonstrated that mesenchymal stem cells (MSCs) can release a large number of functionally specific microRNA (miRNA) microvesicles that play a role in promoting osteogenic differentiation, but the specific mechanism is not yet clear. Under such context, this study aims to elucidate the mechanism of bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exo) promoting fracture healing in mice. We isolated and identified the BMSC-Exo. Bioinformatics analysis predicted high expression of miRNA in exosomes and verified the transfer of miR-25 in exosomes by immunofluorescence. Targeting relationship between miR-25 and Smad ubiquitination regulatory factor-1 (SMURF1) was predicted and verified by dual-luciferase reporter gene assay. Immunoprecipitation and protein stability assays were used to detect Runt-related transcription factor 2 (Runx2) ubiquitination and the effect of SMURF1 on Runx2 ubiquitination, respectively. The effect of miR-25 in BMSC-Exo on fracture healing in mice was assessed using X-ray imaging. alkaline phosphatase, alizarin red staining, EdU, CCK-8, and Transwell were used to evaluate the effects of exosomes transferred miR-25 on osteogenic differentiation, proliferation, and migration of osteoblasts. Bioinformatics analysis predicted that miR-25 expression in exosomes increased significantly. Moreover, the targeted regulation of SMURF1 by miR-25 was verified. SMURF1 inhibited Runx2 protein expression by promoting ubiquitination degradation of Runx2. Notably, miR-25 secreted by BMSC-Exo can accelerate osteogenic differentiation, proliferation, and migration of osteoblasts through SMURF1/Runx2 axis. Our results demonstrate that miR-25 in BMSC-Exo regulates the ubiquitination degradation of Runx2 by SMURF1 to promote fracture healing in mice.

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.

scholarly journals Downregulation of microRNA-149 in retinal ganglion cells suppresses apoptosis through activation of the PI3K/Akt signaling pathway in mice with glaucoma

2018 ◽  
Vol 315 (6) ◽  
pp. C839-C849 ◽  
Author(s):  
Xin-Gang Nie ◽  
Dong-Sheng Fan ◽  
Yan-Xia Huang ◽  
Ying-Ying He ◽  
Bo-Li Dong ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Retinal Ganglion Cells ◽  
Ganglion Cells ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Ultrastructural Changes ◽  
Retinal Ganglion ◽  
Akt Signaling Pathway ◽  
Luciferase Reporter Gene ◽  
Gene Assay

Glaucoma represents a major cause of blindness, generally associated with elevated intraocular pressure (EIOP). The aim of the present study was to investigate whether microRNA-149 (miR-149) affects retinal ganglion cells (RGCs) and the underlying mechanism based on a mouse model of chronic glaucoma with EIOP. The successfully modeled mice were administered with mimics or inhibitors of miR-149. Next, the number of RGCs, ultrastructural changes of RGCs, and purity of RGCs in the retinal tissues were detected. Moreover, the RGCs were collected and subsequently treated with 60 mmHg pressure and transfected with a series of plasmids aiding in the regulation of the expression of miR-149 and betacellulin (BTC). The levels of miR-149, BTC, phosphatidylinositol 3-kinase (PI3K), and Akt were subsequently determined. Finally, RGC viability and apoptosis were detected accordingly. Dual luciferase reporter gene assay provided validation, highlighting BTC was indeed a target gene of miR-149. The downregulation of miR-149 is accompanied by an increased number of RGCs and decreased ultrastructural RGC alterations. Additionally, downregulated miR-149 was noted to increase the levels of BTC, PI3K, and Akt in both the retinal tissues and RGCs, whereas the silencing of miR-149 was observed to promote the viability of RGC and inhibit RGC apoptosis. Taken together, the results of the current study provided validation suggesting that the downregulation of miR-149 confers protection to RGCs by means of activating the PI3K/Akt signaling pathway via upregulation of BTC in mice with glaucoma. Evidence presented indicated the promise of miR-149 inhibition as a potential therapeutic strategy for glaucoma treatment.

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.

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