Downregulation of miR-542-3p promotes osteogenic transition of vascular smooth muscle cells in the aging rat by targeting BMP7

Abstract Background Aging is believed to have a close association with cardiovascular diseases, resulting in various pathological alterations in blood vessels, including vascular cell phenotypic shifts. In aging vessels, the microRNA(miRNA)-mediated mechanism regulating the vascular smooth muscle cell (VSMC) phenotype remains unclarified. MiRNA microarray was used to compare the expressions of miRNAs in VSMCs from old rats (oVSMCs) and young rats (yVSMCs). Quantitative reverse transcription real-time PCR (qRT-PCR) and small RNA transfection were used to explore the miR-542-3p expression in oVSMCs and yVSMCs in vitro. Calcification induction of yVSMCs was conducted by the treatment of β-glycerophosphate (β-GP). Alizarin red staining was used to detect calcium deposition. Western blot and qRT-PCR were used to investigate the expression of the smooth muscle markers, smooth muscle 22α (SM22α) and calponin, and the osteogenic markers, osteopontin (OPN), and runt-related transcription factor 2 (Runx2). Lentivirus was used to overexpress miR-542-3p and bone morphogenetic protein 7 (BMP7) in yVMSCs. Luciferase reporter assay was conducted to identify the target of miR-542-3p. Results Compared with yVSMCs, 28 downregulated and 34 upregulated miRNAs were identified in oVSMCs. It was confirmed by qRT-PCR that oVSMC expressed four times lower miR-542-3p than yVSMCs. Overexpressing miR-542-3p in yVSMCs suppressed the osteogenic differentiation induced by β-GP. Moreover, miR-542-3p targets BMP7 and overexpressing BMP7 in miR-542-3p–expressing yVSMCs reverses miR-542-3p’s inhibition of osteogenic differentiation. Conclusions miR-542-3p regulates osteogenic differentiation of VSMCs through targeting BMP7, suggesting that the downregulation of miR-542-3p in oVSMCs plays a crucial role in osteogenic transition in the aging rat.

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RTEF-1 Inhibits Vascular Smooth Muscle Cell Calcification through Regulating Wnt/β-Catenin Signaling Pathway

Calcified Tissue International ◽

10.1007/s00223-021-00833-4 ◽

2021 ◽

Author(s):

Jingjing Cong ◽

Bei Cheng ◽

Jinyu Liu ◽

Ping He

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Osteogenic Differentiation ◽

Signaling Pathway ◽

Glycogen Synthase ◽

Critical Role ◽

Transcriptional Enhancer ◽

Alizarin Red ◽

Gsk 3Β ◽

Enhancer Factor

AbstractVascular calcification (VC) is highly prevailing in cardiovascular disease, diabetes mellitus, and chronic kidney disease and, when present, is associated with cardiovascular events and mortality. The osteogenic differentiation of vascular smooth muscle cells (VSMCs) is regarded as the foundation for mediating VC. Related transcriptional enhancer factor (RTEF-1), also named as transcriptional enhanced associate domain (TEAD) 4 or transcriptional enhancer factor-3 (TEF-3), is a nuclear transcriptional factor with a potent effect on cardiovascular diseases, apart from its oncogenic role in the canonical Hippo pathway. However, the role and mechanism of RTEF-1 in VC, particularly in calcification of VSMCs, are poorly understood. Our results showed that RTEF-1 was reduced in calcified VSMCs. RTEF-1 significantly ameliorated β-glycerophosphate (β-GP)-induced VSMCs calcification, as detected by alizarin red staining and calcium content assay. Also, RTEF-1 reduced alkaline phosphatase (ALP) activity and decreased expressions of osteoblast markers such as Osteocalcin and Runt-related transcription factor-2 (Runx2), but increased expression of contractile protein, including SM α-actin (α-SMA). Additionally, RTEF-1 inhibited β-GP-activated Wnt/β-catenin pathway which plays a critical role in calcification and osteogenic differentiation of VSMCs. Specifically, RTEF-1 reduced the levels of Wnt3a, p-β-catenin (Ser675), glycogen synthase kinase-3β (GSK-3β), and p-GSK-3β (Ser9), but increased the levels of p-β-catenin (Ser33/37). Also, RTEF-1 increased the ratio of p-β-catenin (Ser33/37) to β-catenin proteins and decreased the ratio of p-GSK-3β (Ser9) to GSK-3β protein. LiCl, a Wnt/β-catenin signaling activator, was observed to reverse the protective effect of RTEF-1 overexpression on VSMCs calcification induced by β-GP. Accordingly, Dickkopf-1 (Dkk1), a Wnt antagonist, attenuated the role of RTEF-1 deficiency in β-GP-induced VSMCs calcification. Taken together, we concluded that RTEF-1 ameliorated β-GP-induced calcification and osteoblastic differentiation of VSMCs by inhibiting Wnt/β-catenin signaling pathway.

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

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02224-w ◽

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.

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Exosomal miR-151-3p Induces Calcium Deposition in Vascular Smooth Muscle Cells by Inhibiting Atg5

10.21203/rs.3.rs-172981/v1 ◽

2021 ◽

Author(s):

Li Chen ◽

Rongrong Zhang ◽

Jinyin Li ◽

Yiping Gao ◽

Shilong Mao

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Calcium Deposition ◽

Alizarin Red ◽

Potential Biomarker

Abstract Background: Calcium deposition in vascular smooth muscle cells (VSMCs) can lead to the rigidity of the vasculature and an increase of risk in cardiac events. This study aimed to explore the role of exosomal microRNA-151-3p (miR-151-3p) in the regulation of VSMC calcification. Methods: A cellular calcification model was established using the mouse primary aortic VSMCs by β-glycerophosphate treatment. The calcium deposition was evaluated by Alizarin Red staining. The expression of miR-151-3p in exosomes was evaluated by qRT-PCR. The relationship between miR-151-3p and Atg5 was determined by bioinformatics analysis and dual-luciferase gene reporter assay. The exosome derived from mouse VSMCs transfected with miR-151-3p mimics/inhibitor were isolated and used to stimulate VSMCs. The expression of Atg5, α-SMA, OPN, Runx2 and BMP2 was evaluated by western blot. An animal model was established to investigate the role of miR-151-3p in exosomes.Results: MiR-151-3p was significantly upregulated in the exosomes of VSMCs treated with β-glycerophosphate. Exosomes derived from calcific VSMCs increased the calcium deposition of general VSMCs without any treatment. Exosomes derived from miR-151-3p mimics transfected VSMCs increased the expression of Runx2 and BMP2, while reduced the expression of α-SMA and OPN in general VSMCs. and exosomes derived from miR-151-3p inhibitor transfected VSMCs reversed these effects in vitro. Meanwhile, miR-151-3p served as a ceRNA of Atg5 by directly binding to the 3'UTR of Atg5. Moreover, the expression of α-SMA, OPN, Runx2 and BMP2 in vivo was consistent with the results in VSMCs in vitro.Conclusion: Our study revealed that miR-151-3p in VSMCs-derived exosomes might induce calcium deposition through regulating Atg5 expression, suggesting that miR-151-3p might be a potential biomarker for vascular calcification.

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

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.648627 ◽

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.

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MO441CALPROTECTIN IS A NOVEL CONTRIBUTING FACTOR IN VASCULAR CALCIFICATION AND A PREDICTOR OF CARDIOVASCULAR OUTCOME IN CKD PATIENTS*

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfab090.003 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

Ana Amaya Garrido ◽

José M Valdivielso ◽

Stanislas Faguer ◽

Arnaud Del Bello ◽

Benedicte Buffin-Meyer ◽

...

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Vascular Calcification ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Calcium Deposition ◽

Alizarin Red ◽

Serum Proteome ◽

Ckd Stage

Abstract Background and Aims Vascular calcification, leading to aortic stiffening and heart failure, is decisive risk factor for cardiovascular (CV) mortality in patients with chronic kidney disease (CKD). Promoted by bone mineral disorder and systemic inflammation in CKD patients, vascular calcification is a complex mechanism involving osteochondrogenic differentiation of vascular smooth muscle cells (VSMCs) and abnormal deposition of minerals in the vascular wall. Despite intensive research efforts in recent years, available treatments have limited effect and none of them prevent or reverse vascular calcification. The aim of this study was to analyse the serum proteome of CKD stage 3-4 patients in order to unravel new molecular changes associated to CV morbid-mortality and to decipher the role of novel candidates on vascular calcification to provide potential new therapeutic agents. Method In this study we used serum samples from two independent cohorts: 112 CKD stage 3-4 patients with a 4 years follow-up for CV events and 222 CKD stage 5 patients exhibiting a broad range of calcification degree determined by histological quantification in the epigastric and/or iliac artery. Serum proteome analysis was performed using tandem mass-spectrometry in a subcohort of 66 CKD3-4 patients and validation of protein candidates was performed using ELISA in the two full cohorts. Human primary vascular smooth muscle cells and mouse aortic rings were used for calcification assays. Calcium content was quantified using QuantiChrom calcium assay kit and calcium deposition was visualized by Alizarin Red and Von Kossa staining. Results Among 443 proteins detected in the serum of CKD3-4 patients, 134 displayed significant modified abundance in patients with CV events (n=32) compared to patients without (n=34). One of the most prominent changes was increased level of calprotectin (up to 8.6 fold, P<.0001). Using ELISA, we validated that higher serum calprotectin levels were strongly associated with higher probability of developing CV complications and increased mortality in CKD stage 3-4 patients (Figure A). Moreover, we showed that higher serum calprotectin was associated with increased vascular calcification levels in CKD stage 5 patients (Figure B). In vitro, calprotectin promoted calcification of human VSMCs (p<0.0001) (Figures C-D) and in mouse aortic rings (p<0.0001) (Figure E-F). Interestingly, these effects were significantly attenuated by paquinimod, a calprotectin inhibitor (Figures C-F). Conclusion Circulating calprotectin is a novel predictor of CV outcome and mortality in CKD patients. Calprotectin also shows calcification-inducing properties and its blockade by paquinimod alleviates its effects. Future experiments will consist in deciphering the signalling pathways involved in the regulation of calcification by calprotectin and evaluating in vivo the therapeutic potential of paquinimod on the development of medial vascular calcification lesions associated with CKD.

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Melatonin Promotes Bone Marrow Mesenchymal Stem Cell Osteogenic Differentiation and Prevents Osteoporosis Development Through Modulating circ_0003865 that Sponges miR-3653-3p

10.21203/rs.3.rs-50665/v1 ◽

2020 ◽

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: To investigate circRNAs in Melatonin (MEL)-regulated bone marrow mesenchymal stem cell (BMSC) differentiation and osteoporosis.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 were validated for the osteogenic differentiation of BMSCs by qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on osteoporosis (OP) development was tested in murine osteoporosis 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 promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSCs osteogenic differentiation induced by MEL. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing. Finally, circ_0003865 silencing repressed OP development in mouse model.Conclusion: MEL promotes BMSC osteogenic differentiation and inhibits osteoporosis pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

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Thymoquinone loading into hydroxyapatite/alginate scaffolds accelerated the osteogenic differentiation of the mesenchymal stem cells

10.21203/rs.3.rs-467670/v1 ◽

2021 ◽

Author(s):

Ebrahim Rahmani-Moghadam ◽

Tahereh Talaei-Khozani ◽

Vahideh Zarrin ◽

Zahra Vojdani

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Osteogenic Differentiation ◽

Physical And Mechanical Properties ◽

Calcium Deposition ◽

Alizarin Red ◽

Qrt Pcr ◽

Alp Activity ◽

Sintering Method

Abstract Background: Hydroxyapatite (HA) can be loaded by some osteogenic inducing agents such as thymoquinone (TQ) and alginate. This study was performed to investigate the effect of TQ loading into HA/alginate scaffolds on osteogenic differentiation capability of mesenchymal stem cells.Methods: HA scaffolds were fabricated by casting and sintering method and impregnated by TQ containing alginate. The stem cells were loaded onto the scaffolds and induced to differentiate into osteoblasts. Alkaline Phosphatase (ALP) activity, Alizarin Red S, Real-Time qRT-PCR, and MTT assessments were done. Finally, the cells were examined with a light microscope, confocal microscope, and SEM.Results: The results showed that the presence of the alginate decelerates the degradation rate and reinforces the mechanical strength. while the presence of TQ had no significant influence on physical and mechanical properties of the HA/alginate scaffolds, it led to a significant increase in ALP activity and expression of collagen, osteopontin, and osteocalcin at early phase of differentiation. Also, TQ administration had no impact on calcium deposition and proliferation as well as bone-marker expression at long term differentiation.Conclusion: TQ accelerates the differentiation of the stem cells into the osteoblasts without changing the properties of the scaffolds, and the HA/alginate/TQ scaffold can be used as a scaffold with osteogenic properties in bone tissue engineering applications.

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Effect of MicroRNA-26a Targeting mCTGF on Calcification of Vascular Smooth Muscle Cells Through Regulating Osteoprotegerin/Receptor Activator of NF-kB/Receptor Activator of Nuclear Factor Kappa-B Ligand Signaling Pathway

Journal of Biomaterials and Tissue Engineering ◽

10.1166/jbt.2020.2516 ◽

2020 ◽

Vol 10 (12) ◽

pp. 1813-1819

Author(s):

Zhe Zhang ◽

Jing Cheng ◽

Zhen Yang

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Smooth Muscle Cells ◽

Signaling Pathway ◽

Vascular Smooth Muscle Cells ◽

Muscle Cells ◽

Normal Group ◽

Luciferase Reporter ◽

Qrt Pcr ◽

Receptor Activator

To explore the effect of microRNA-26a targeting mCTGF on inhibiting the calcification of vascular smooth muscle cells (VSMCs) through OPG/RANK/RANKL signaling pathway. VSMCs were isolated and cultured by adherent method. The morphology of VSMCs was observed under microscope and the surface antigen was identified by flow cytometry. The calcification of VSMCs was detected using alizarin red S staining and oil red O staining. microRNA-26a level in supernatant of cultured VSMCs was detected by ultracentrifugation method. The binding site between microRNA-26a and 3′-UTR of mCTGF was predicted by bioinformatics software; VSMCs were induced to be calcified and verified by the luciferase reporter gene assay. The mRNA and protein expressions of OPG/RANK/RANKL signaling pathway were measured by qRT-PCR and Western blotting. Normal VSMCs were assigned into normal group (normal VSMCs were treated with the exosomes secreted by normal VSMCs), CKD group (normal VSMCs were treated with the exosomes secreted by VSMCs in mice with chronic kidney disease (CKD)) and CKD/overexpressed mCTGF group (normal VSMCs were treated with the exosomes secreted by VSMCs in CKD mice and meanwhile treated with overexpressed mCTGF) followed by analysis of OPG/RANK/RANKL signaling level by qRT-PCR and ALP activity. VSMCs grew against the wall and showed a long fusiform shape. Calcification and adipogenesis of VSMCs was found under different induction conditions. microRNA-26a level in the supernatant of VSMCs in CKD group was significantly higher than normal group; after microRNA-26a bound to of mCTGF, mCTGF level in the CKD/overexpressed mCTGF group was significantly lower. The expression of OPG/RANK/RANKL signaling pathway was significantly decreased in the CKD/overexpressed mCTGF group. Meanwhile, OPG/RANK/RANKL signaling protein was increased significantly after treatment of exosomes secreted by VSMCs in CKD mice. mCTGF is the target gene of microRNA-26a and microRNA-26a can target mCTGF to regulate exosomes secretion by VSMCs and OPG/RANK/RANKL signaling to inhibit the calcification of VSMCs.

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The Bioactive Substance Secreted by MSC Retards Mouse Aortic Vascular Smooth Muscle Cells Calcification

BioMed Research International ◽

10.1155/2018/6053567 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10 ◽

Cited By ~ 3

Author(s):

Shuangshuang Wang ◽

Maoqing Tong ◽

Siwang Hu ◽

Xiaomin Chen

Keyword(s):

Smooth Muscle ◽

Vascular Smooth Muscle ◽

Intracellular Calcium ◽

Vascular Calcification ◽

Calcium Content ◽

Calcium Deposition ◽

Expression Ratio ◽

Alizarin Red ◽

Alp Activity ◽

Study Results

Background. Vascular calcification, which is associated with low-level chronic inflammation, is a complication that occurs during aging, atherosclerosis, chronic kidney disease, diabetes mellitus, and hyperlipaemia. In this study, we used conditioned media from mesenchymal stem cells (MSC-CM), a source of autologous cytokines, to test the hypothesis that MSC-CM inhibits vascular smooth muscle cell (VSMC) calcification by suppressing inflammation and apoptosis. Methods. VSMCs were treated with β-glycerophosphate (β-GP) to induce calcification and MSC-CM was used as a treatment. Calcium deposition was evaluated using alizarin red and von Kossa staining after a 7-day induction period. Intracellular calcium contents were measured via the o-cresolphthalein complexone method, and alkaline phosphatase (ALP) activity was determined using the para-nitrophenyl phosphate method. The expressions of specific-osteogenic markers, inflammatory cytokines, and apoptosis-associated genes/proteins were examined by real-time polymerase chain reaction or western blotting. Results. MSC-CM inhibited β-GP-induced calcium deposition in VSMCs and decreased intracellular calcium content and ALP activity. Additionally, MSC-CM suppressed the β-GP-induced increases in BMP2, Msx2, Runx2, and osteocalcin expression. Additionally, MSC-CM decreased the expression of TNF-α, IL-1β, and IL-6 in VSMC. MSC-CM also partly blocked β-GP-induced VSMC apoptosis, which was associated with an increase in the Bcl-2/Bax expression ratio and a decrease in caspase-3 expression. Conclusion. Our study results suggest that MSC-CM can inhibit VSMC calcification. This suggests a potential novel clinical application for MSCs in the treatment of vascular calcification and associated diseases.

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

10.21203/rs.3.rs-50665/v2 ◽

2020 ◽

Author(s):

Xudong Wang ◽

Taiqiu Chen ◽

Zhihuai Deng ◽

Wenjie Gao ◽

Tongzhou Liang ◽

...

Keyword(s):

Bone Marrow ◽

Osteogenic Differentiation ◽

Luciferase Reporter ◽

Specific Gene ◽

Biological Processes ◽

Rt Pcr ◽

Alizarin Red ◽

Qrt Pcr ◽

Marrow Mesenchymal Stem Cells ◽

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 cells (BMSCs) osteogenic differentiation and osteoporosis progression.The aim of our study was to investigate circRNAs in MEL-regulated BMSCs differentiation and osteoporosis progression.Methods: BMSCs 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 were validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on osteoporosis (OP) development was tested in murine osteoporosis 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 BMSCs 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. BMSCs osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSCs proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSCs 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 BMSCs osteogenic differentiation and inhibits osteoporosis pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

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