Effect of Mir-1301 on the Proliferation and Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Hormone Osteonecrosis via Regulating SRY Related High Mobility Group Box 11 (SOX-11)

2020 ◽  
Vol 10 (12) ◽  
pp. 1858-1864
Author(s):  
Bingshen Jia ◽  
Guoxin Qu ◽  
Peng Yu ◽  
Tuo Jiao ◽  
ZiZhenbiao Wang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
High Mobility ◽  
Hormone Treatment ◽  
Widespread Application ◽  
Runx2 Expression ◽  
Promote Cell Proliferation ◽  
Alp Activity

The widespread application of hormones leads to steroidal osteonecrosis and BMSCs have important roles in treating steroidal osteonecrosis. Mir-1301 involves in several diseases. However, Mir-1301’s effect on BMSCs proliferation and osteogenic differentiation in hormonal osteonecrosis has not been elucidated. Rat BMSCs were isolated and assigned into control groups; Dex group (1μM dexamethasone was added); Mir-1301 group and si-Mir-1301 group followed by analysis of miR-1301 and SOX11 level by Real time PCR, cell proliferation by MTT assay, Caspase3 activity kit, OPN and Runx2 expression by Real time PCR, and ALP activity. Under hormone treatment, Mir-1301 expression in BMSCs cells was significantly increased, proliferation was inhibited, Caspase3 activity was increased, SOX11, OPN and Runx2 expression was decreased and ALP activity was reduced (P <0.05). The above changes were more significant after transfection of Mir-1301 mimics (P <0.05). The addition of Mir-1301 inhibitor to Dex-treated BMSCs could down-regulate Mir-1301, significantly increase the expression of SOX11, OPN and Runx2, promote cell proliferation, decrease Caspase3 activity and increase ALP activity (P <0.05). The target gene of Mir-1301 is SOX11. Mir-1301 expression in BMSCs cells is increased during steroid-induced osteonecrosis. Down-regulating Mir-1301 during steroid-induced osteonecrosis can inhibit BMSCs apoptosis and promote proliferation and osteogenesis via targeting SOX11.

Effect of αCGRP on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through Smad/Runx2 Signaling Pathway

2020 ◽  
Vol 10 (6) ◽  
pp. 868-873
Author(s):  
Shengxiang Huang ◽  
Haibo Mei ◽  
Rongguo He ◽  
Kun Liu ◽  
Jin Tang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

The α-calcitonin gene-related peptide (α-CGRP) regulates bone metabolism and has potential applications in enhancing bone remodeling in vivo. However, α-CGRP's role in bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation remain unclear. Rat BMSCs were separated into control group, α-CGRP group and α-CGRP siRNA group, in which BMSCs were transfected with α-CGRP plasmid and α-CGRP siRNA respectively followed by analysis of α-CGRP level by real time PCR and ELISA, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, formation of calcified nodules by alizarin red staining, Smad1 and Smad7 level by Western blot and Runx2 by real time PCR. αCGRP transfection into BMSCs significantly up-regulated CGRP, which could promote cell proliferation, inhibit Caspase 3 activity, promote ALP activity, increase calcified nodules formation and upregulate Smad1, Smad7 and Runx2 compared to control (P < 0.05); transfection of αCGRP siRNA significantly down-regulated CGRP in BMSCs, inhibited cell proliferation, promoted Caspase 3 activity, inhibited ALP activity, inhibited calcified nodules formation and downregulate Smad1, Smad7 and Runx2 (P < 0.05). αCGRP overexpression promotes the Smad/Runx2 signaling, which in turn promotes BMSCs proliferation and osteogenesis. Decreased αCGRP level inhibits Smad/Runx2 signaling, promotes BMSCs apoptosis, inhibits proliferation and osteogenic differentiation.

Effect of SOX9 on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Through WNTβ/Catenin Pathway

2019 ◽  
Vol 9 (10) ◽  
pp. 1429-1434
Author(s):  
Qing Yang ◽  
Cheng Li ◽  
Manli Yan ◽  
Chunhua Fang
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Proliferation ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells

Bone marrow mesenchymal stem cells (BMSCs) can be differentiated into different types of cells. SOX9 involves in the development and progression of various diseases. Our study aims to assess SOX9's effect on osteogenic differentiation of BMSCs and its related regulatory mechanisms. Rat BMSCs were isolated and randomly divided into control group, SOX9 group and SOX9 siRNA group, which was transfected with pcDNA-SOX9 plasmid or SOX9 siRNA respectively followed by analysis of SOX9 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 and ALP activity, GSK-3β expression and Wntβ/Catenin Signaling pathway protein expression by Western blot, and expression of osteogenic genes Runx2 and BMP-2 by Real time PCR. Transfection of pcDNA-SOX9 plasmid into BMSCs significantly inhibited cell proliferation, promoted Caspase3 activity, decreased ALP activity and downregulated Runx2 and BMP-2, increased GSK-3β expression and decreased Wntβ/Catenin expression protein expression (P< 0.05). SOX9 siRNA transfection significantly promoted cell proliferation, inhibited Caspase3 activity, increased ALP activity and upregulated Runx2 and BMP-2, downregulated GSK-3β and increased Wntβ/Catenin expression. SOX9 regulates BMSCs proliferation and osteogenic differentiation through Wntβ/Catenin signaling pathway.

Transforming Growth Factor Beta (TGF-β) Regulates Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells by Promoting Wnt Signaling Pathway in Atrophic Nonunion

2020 ◽  
Vol 10 (2) ◽  
pp. 265-270
Author(s):  
Minqing Zhan ◽  
Mingming Wang ◽  
Juan Zhang ◽  
Xiaorui Jiang
Keyword(s):  
Cell Proliferation ◽  
Wnt Signaling ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Wnt Signaling Pathway ◽  
The Body ◽  
Alp Activity ◽  
Atrophic Nonunion

During atrophic nonunion, Wnt signaling pathway is inhibited, resulting in inhibition of BMSC osteogenic differentiation. TGF-β regulates growth and development of the body. However, TGF-β’s effect on osteogenic differentiation of BMSCs in atrophic nonunion has not been reported. The bone tissue and serum of patients with atrophic nonunion and normal healing fractures were collected, and TGF-β mRNA and serum secretion were analyzed by Real time PCR and ELISA. Rat BMSCs were cultured and randomly divided into control group, TGF-β group and TGF-β siRNA group which was transfected with pcDNA-TGF-β plasmid or TGF-β siRNA respectively followed by analysis of cell proliferation by MTT assay, alkaline phosphatase (ALP) activity, Caspase3 activity, expression of RUNX2 and OPN and PPARγ2 mRNA by Real time PCR, and WNT5A and FZD3 expression by Western blot. TGF-β mRNA level and secretion in patients with atrophic nonunion was significantly reduced compared with patients with normal healing fractures (P < 0.05). Transfection of TGF-β siRNA down-regulated TGF-β expression in BMSCs, significantly inhibited cell proliferation, increased Caspase3 activity, decreased ALP activity, RUNX2 and OPN expression, increased PPARγ2 expression and deceased WNT5A and FZD3 expression (P < 0.05). However, transfection of pcDNA-TGF-β plasmid up-regulated TGF-β expression in BMSCs and reversed the above changes (P < 0.05). TGF-β is reduced in atrophic nonunion patients. Targeting TGF-β promotes BMSCs proliferation and osteogenic differentiation by regulating Wnt signaling pathway.

Down-Regulation of Notch-2 Promotes the Osteogenic Differentiation of Bone Marrow Stromal Cells in High Glucose Environment by Targeting Fat Mass and Obesity-Associated Protein

2019 ◽  
Vol 9 (10) ◽  
pp. 1459-1465
Author(s):  
Zhenhua Zhang ◽  
Dianguo Li ◽  
Xiaogang Sun ◽  
Jialong Xu
Keyword(s):  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
High Glucose ◽  
Real Time Pcr ◽  
Caspase 3 ◽  
Down Regulation ◽  
Sod Activity ◽  
Alp Activity ◽  
High Glucose Group

BMSCs Differentiation into osteoblasts is beneficial for treating osteoporosis. Notch signaling pathway regulates the proliferation of BMSCs. However, Notch-2's effect on osteogenic differentiation of BMSCs in high glucose environment remains unclear. Rat BMSCs were isolated and divided into control group, high glucose group, Notch-2 siRNA group, in which BMSCs cells were transfected with Notch-2 siRNA under high glucose environment followed by analysis of Notch-2 mRNA expression by Real time PCR, cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and BMP-2 by Real time PCR, TNF-α and IL-2 secretion by ELISA, myeloperoxidase (MPO) and superoxide dismutase (SOD) activity and FTO protein expression by Western blot. In high glucose group, Notch-2 expression was increased with inhibited cell proliferation, increased Caspase 3 activity and decreased ALP activity. Meanwhile, high glucose significantly increased MPO content, decreased SOD activity, increased TNF-α and IL-2 secretion and FTO expression, and reduced Runx2 and BMP-2 expression (P < 0.05); Notch-2 siRNA transfection significantly down-regulated Notch-2 expression, promoted BMSCs cell proliferation, decreased Caspase 3 activity and increased ALP activity. In addition, it also significantly decreased MPO content, increased SOD activity, decreased TNF-α and IL-2 and FTO expression as well as increased Runx2 and BMP-2 expression (P < 0.05). Notch-2 is upregulated in BMSCs under high glucose. Down-regulation of Notch-2 in BMSCs under high glucose environment could promote BMSCs proliferation and osteogenic differentiation.

The Regulatory Effect of Mir-149 on Bone Marrow Mesenchymal Stem Cells in Osteoporosis Rats and Its Related Mechanisms

2019 ◽  
Vol 9 (8) ◽  
pp. 1127-1132 ◽  
Author(s):  
Long Wang ◽  
Jian Yu
Keyword(s):  
Cell Proliferation ◽  
Real Time ◽  
Real Time Pcr ◽  
Mapk Signaling ◽  
Control Group ◽  
Regulatory Effect ◽  
Erk Mapk ◽  
Alp Activity ◽  
Sd Rats ◽  
Marrow Mesenchymal Stem Cells

BMSCs play an important role in osteoporosis (OP) and their differentiation can lead to OP progression. Mir-149 can participate in the regulation of BMSCs. However, the effect of Mir-149 on BMSCs in osteoporosis remains unclear. SD rats were divided control group and OP group. OP rat BMSCs were transfected with Mir-149 siRNA followed by analysis of Mir-149 expression by real time PCR, cell proliferation by MTT assay, apoptosis by flow cytometry, ERK/MAPK signaling protein expression by western blot, ALP activity, as well as expression of osteogenic genes Runx2 and OC by real time PCR. Mir-149 expression was significantly increased in BMSCs of OP rats, cell proliferation was inhibited, apoptosis was increased, as well as p-ERK1/2 expression, ALP activity and expression of Runx2 and OC was decreased compared to control group (P < 0.05). Transfection of Mir-149 siRNA into OP rat BMSCs reduced Mir-149 expression, promoted cell proliferation, decreased apoptotic rate, increased p-ERK1/2 expression, ALP activity and Runx2 and OC expression. Compared with OP group, the differences were statistically significant (P< 0.05). Mir-149 expression was increased in OP rat BMSCs. Down-regulation of Mir-149 promoted the activation of ERK/MAPK signaling pathway, inhibited apoptosis of BMSCs and promoted the proliferation and osteogenic differentiation of BMSCs in OP rats.

Effect of Silent Information Regulator on the Survival and Osteogenic Differentiation of Inflammatory Bone Marrow Mesenchymal Stem Cells by Regulating NF-κB Signaling Pathway

2020 ◽  
Vol 10 (1) ◽  
pp. 121-126
Author(s):  
Wenkun Lu ◽  
Tao Wang ◽  
Xunjian Gao ◽  
Fuqiang Yang ◽  
Jianjian Ge
Keyword(s):  
Cell Proliferation ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Caspase 3 ◽  
Adipogenic Differentiation ◽  
Control Group ◽  
Promote Cell Proliferation ◽  
Alp Activity ◽  
Marrow Mesenchymal Stem Cells ◽  
Inflammation Group

Osteogenic differentiation of BMSCs is beneficial for osteoarthritis (OA) treatment. Silent information regulator (SIRT1) plays a role in endocrine diseases and aging-related diseases. However, the role of SIRT1 in OA has not yet been elucidated. Rat BMSCs were isolated and divided into control group, inflammation group (BMSCs were cultured with IL-6), SIRT1 group (SIRT1 agonist Resveratrol was added under the action of IL-6) followed by analysis of cell proliferation by MTT assay, Caspase 3 activity, ALP activity, expression of osteogenic genes Runx2 and OC and adipogenic differentiation gene PPARγ2 by Real time PCR, NF-κB expression by western blot and secretion of TNF-α and IL-6 by ELISA. In inflammation group, SIRT1 expression was significantly decreased, cell proliferation was significantly inhibited, and Caspase 3 activity was increased. Meanwhile, ALP activity, Runx2 and OC expression was decreased, PPARγ2 and NF-κB expression was increased, along with elevated TNF-α and IL-6 secretion compared to control (P < 0.05). Resveratrol can significantly promote the expression of SIRT1 in BMSCs of inflammation group, promote cell proliferation, decrease Caspase 3 activity, and increase Runx2 and OC expression. In addition, it decreased PPARγ2 and NF-κB expression and reduced the secretion of TNF-α and IL-6 (P < 0.05). The expression of SIRT1 was decreased in BMSCs under inflammation. SIRT1 overexpression in BMSCs under inflammation inhibits inflammation, promotes proliferation and osteogenic differentiation of BMSCs through regulating NF-κB signaling pathway.

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.

Effect of Long-Chain Non-Coding RNA GAS5 on Osteogenic/Adipogenic Differentiation of Bone Marrow Mesenchymal Stem Cells Under Oxidative Stress Through Targeting MiR-365

2019 ◽  
Vol 9 (12) ◽  
pp. 1751-1757
Author(s):  
Wenming Wu ◽  
Dongming Liang
Keyword(s):  
Oxidative Stress ◽  
Cell Proliferation ◽  
Real Time ◽  
Real Time Pcr ◽  
Adipogenic Differentiation ◽  
Control Group ◽  
Sod Activity ◽  
Stress Group ◽  
Alp Activity ◽  
Lncrna Gas5

Oxidative stress affects BMSCs. LncRNA GAS5 regulates cell proliferation and apoptosis. However, the effect of LncRNA GAS5 on osteogenesis/adipogenic differentiation of BMSCs under oxidative stress has not been reported. Rat BMSCs were cultured and randomly divided into 4 groups, normal control group; oxidative stress group; GAS5 siRNA group; GAS5 siRNA+ miR-365 inhibitor group followed by analysis of LncRNA GAS5 expression by Real time PCR, cell proliferation by MTT assay, Caspase3 activity, GAS5 and miR-365 targeting relationship by luciferase reporter assay, ALP activity, expression of Runx2, OP and PPAR 2 by Real time PCR, as well as ROS content and SOD activity. In oxidative stress group, GAS5 expression was significantly increased along with inhibited cell proliferation, increased Caspase3 activity, decreased ALP activity and the expression of Runx2 and OP, increased PPAR 2 expression and ROS content, and decreased SOD activity compared to control group (P < 0 05). miR-365 was the target miRNA of GAS5. GAS5 siRNA down-regulated GAS5 expression, significantly promoted cell proliferation, inhibited Caspase3 activity, increased ALP activity and Runx2 and OP expression, decreased PPAR 2 expression and ROS content, and increased SOD activity. (P < 0 05). However, GAS5 siRNA+ miR-365 inhibitor group reversed the effect of GAS5 siRNA. Oxidative stress promotes LncRNA GAS5 expression in BMSCs. LncRNA GAS5 regulates oxidative stress by targeting miR-365. Knockdown of GAS5 can promote BMSCs proliferation and osteogenic differentiation and inhibit adipogenic differentiation under oxidative stress.

Overexpression of Heme Oxygenase-1 Promotes Osteoblast Differentiation of Osteoporosis Rat Bone Marrow Stromal Cells in Bone Morphogenetic Protein-Dependent Manner

2019 ◽  
Vol 9 (11) ◽  
pp. 1614-1620
Author(s):  
Jiangrong Fan ◽  
Yong Zheng ◽  
Jingyang You
Keyword(s):  
Cell Proliferation ◽  
Real Time ◽  
Heme Oxygenase ◽  
Real Time Pcr ◽  
Osteoblast Differentiation ◽  
Caspase 3 ◽  
Control Group ◽  
Heme Oxygenase 1 ◽  
Dependent Manner ◽  
Alp Activity

BMSCs play a role in osteoporosis (OP) and their differentiation can lead to OP progression. Heme oxygenase-1 (HO-1) involves in many diseases, but the effect of HO-1 on osteoblast differentiation of BMSCs in OP rats remains unclear. SD rats were divided into control group and OP group. Rats BMSCs in OP group were cultured in vitro, HO-1 expression was up-regulated by HO-1 agonist hemin, and BMPR inhibitor LDN-19318 was added followed by analysis of HO-1 expression by real time PCR and ELISA, cell proliferation by MTT assay, apoptosis by Caspase 3 activity, BMP-2 expression by Western blot, ALP activity, expression of Runx2 and OC by real time PCR. In OP group, HO-1 expression was significantly decreased, cell proliferation was inhibited, Caspase 3 activity was increased along with decreased ALP activity and expression of Runx2, OC and BMP-2 compared to control (P < 0.05). Up-regulation of HO-1 expression significantly promoted cell proliferation, reduced Caspase 3 activity, increased ALP activity, and expression of Runx2, OC and BMP-2 (P < 0.05). However, inhibition of HO-1 significantly promoted bone differentiation after the addition of BMPR inhibitor LDN-193189 (P < 0.05). HO-1 expression is decreased in BMSCs of OP group rats. Up-regulation of HO-1 promoted BMSCs proliferation in OP rats in BMP-dependent manner, inhibited apoptosis, and promoted osteoblast differentiation.

scholarly journals MiR-145 regulates osteogenic differentiation of human adipose-derived mesenchymal stem cells through targeting FoxO1

2017 ◽  
Vol 243 (4) ◽  
pp. 386-393 ◽  
Author(s):  
Wei Hao ◽  
Hongzhi Liu ◽  
Lugang Zhou ◽  
Yujie Sun ◽  
Hao Su ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Real Time Pcr ◽  
Immunofluorescence Assay ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Alp Activity ◽  
Gene Assay ◽  
Before And After

In this study, we aimed to investigate the expression of miR-145 before and after hASCs osteogenic differentiation. We also intended to explore the influence of the target relationship between miR-145 and FoxO1 on osteogenic differentiation. Dual-luciferase reporter gene assay and real-time PCR were used to confirm the target relationship between miR-145 and FoxO1. Furthermore, the modulatory effects of miR-145 and FoxO1 on hASCs osteoinductive differentiation were measured by real-time PCR , Western blot, ALP staining, ARS staining, and cell immunofluorescence assay. After osteogenic differentiation, miR-145 was gradually down-regulated, while FoxO1 was up-regulated in hASCs. MiR-145 could directly target FoxO1 3′UTR. FoxO1 was negatively regulated by miR-145. After osteoinductive differentiation, BSP, Ocn, and OPN expression was lowered with the overexpression of miR-145 or the knockdown of FoxO1. Furthermore, ALP and ARS staining assay results showed weakened ALP activity and extracellular matrix calcification. When overexpressing miR-145 and FoxO1 simultaneously, no obvious change in ALP activity and extracellular matrix calcification was seen. MiR-145 could suppress hASCs osteoinductive differentiation by suppressing FoxO1 directly. Impact statement Researching on ASCs was a promising strategy to study osteogenic differentiation. The regulatory role of miR-145 on hASCs osteogenic differentiation remained partially explored. Our study revealed a novel mechanism of the osteogenic differentiation process and suggested that miR-145 and its target gene FoxO1 may be potential targets for the therapy of human osteogenic-related disorders.

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