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.

scholarly journals N-CoR modulates osteogenic differentiation of rat mesenchymal stem cells through the PI3K/Akt-cell signaling pathway

2016 ◽  
Vol 68 (3) ◽  
pp. 551-559
Author(s):  
Yi Qin ◽  
Guoping Cao ◽  
Jichao Ye ◽  
Peng Wang ◽  
Liangbin Gao ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Protein Expression ◽  
Osteogenic Differentiation ◽  
Mrna Expression ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Akt Pathway ◽  
Akt Signaling Pathway ◽  
Alp Activity

The nuclear receptor corepressor (N-CoR) is involved in the regulation of diverse transcription factors. We previously found that N-CoR could regulate adipogenic differentiation of rat mesenchymal stem cells (MSCs),but whether it modulated osteogenic differentiation of this type of cells was uncertain. Therefore, in the present study, we investigated the effect and mechanism of N-CoRon osteogenic differentiation of rat MSCs. The results showed that MSCs cultured in osteogenic medium successfully differentiated into osteogenic cells. Overexpression of N-CoR decreased cell proliferation, alkaline phosphatase (ALP)activity, calcium accumulation, mRNA expression of genes including bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN), Osterix and Runx2, and protein expression of phosphor-Akt(pAkt). Conversely, knocking down cellular N-CoR by small interfering RNA (siRNA) promoted pAkt activity and cell differentiation. Overexpression or knockdown of N-CoRhad no significant influences on the protein expression of pyruvate dehydrogenase kinase isozyme 1 (PDK1), phosphatidylinositol 3-kinase (PI3K) and total Akt, indicating that N-CoR regulated the changes in the PI3K/Akt signaling pathway by targeting pAkt. To further prove the function of the PI3K/Akt signaling in N-CoR-regulated osteogenic differentiation, we used the PI3K inhibitor (LY294002) to block the activation of the PI3K/Akt pathway and found that overexpression of N-CoR showed no effects on ALP activity, calcium level and mRNA expression of BSP, osteocalcin OCN, OPN, Osterix and Runx2 in rat MSCs following the inhibition of the PI3K/Akt pathway. These findings suggest that N-CoR regulates osteogenic differentiation of rat MSCs through suppression of the PI3K/Akt signaling pathway.

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 MicroRNA-331 Inhibits Proliferation and Invasion of Melanoma Cells by Targeting Astrocyte-Elevated Gene-1

2018 ◽  
Vol 26 (9) ◽  
pp. 1429-1437 ◽  
Author(s):  
Li Chen ◽  
Guozhang Ma ◽  
Xiaohui Cao ◽  
Xiaoxia An ◽  
Xiguang Liu
Keyword(s):  
Signaling Pathway ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression Patterns ◽  
Polymerase Chain Reaction Analysis ◽  
Melanoma Cells ◽  
Akt Signaling ◽  
Chemotherapeutic Agents ◽  
Luciferase Reporter ◽  
Akt Signaling Pathway ◽  
Proliferation And Invasion

Melanoma is characterized by aggressive invasion, early metastasis, and resistance to existing chemotherapeutic agents. Accumulated studies have reported that microRNA (miRNA) is a potentially robust molecular tool for developing future therapeutic technologies. Therefore, examining the expression patterns, biological roles, and associated mechanisms of cancer-related miRNAs in melanoma is essential for developing novel therapeutic targets for patients with this disease. In this study, miRNA-331 (miR-331) was underexpressed in melanoma tissues and cell lines. Functional assays revealed that the enforced expression of miR-331 inhibited cell proliferation and invasion. In addition, astrocyte-elevated gene-1 (AEG-1) was identified as a novel target of miR-331 through bioinformatics analysis, reverse transcription quantitative polymerase chain reaction analysis, Western blot analysis, dual-luciferase reporter assay, and Spearman’s correlation analysis. Furthermore, reintroduction of AEG-1 partially abrogated the inhibitory effects of miR-331 overexpression on the proliferation and invasion of melanoma cells. Moreover, miR-331 suppressed the activation of the PTEN/AKT signaling pathway in melanoma by inhibiting AEG-1. In short, miR-331 may play tumor-suppressive roles in melanoma by directly targeting AEG-1 and regulating the PTEN/AKT signaling pathway, suggesting that miR-331 could be investigated as a therapeutic strategy for patients with this malignancy.

scholarly journals MicroRNA-26a inhibits wound healing through decreased keratinocytes migration by regulating ITGA5 through PI3K/AKT signaling pathway

2020 ◽  
Vol 40 (9) ◽  
Author(s):  
Zhongping Jiang ◽  
Jie Wei ◽  
Weize Yang ◽  
Wen Li ◽  
Feng Liu ◽  
...  
Keyword(s):  
Wound Healing ◽  
Protein Expression ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Luciferase Reporter ◽  
Epidermal Keratinocytes ◽  
Dependent Manner ◽  
Hacat Cells ◽  
Akt Signaling Pathway ◽  
Tgf Β1

Abstract Background: Keratinocyte migration is essential for skin wound healing and recent studies demonstrated that microRNAs (miRNAs) are involved in the differentiation, migration and apoptosis in keratinocytes. However, the function of miR-26a in wound healing remains to be largely explored. Methods: Northern blot and quantitative reverse transcriptase PCR (qRT-PCR) were used to detect the miR-26a expression and Western blot was used to detect integrin α-5 (ITGA5), phosphatidylinositol-3-kinase (PI3K), p-PI3K, protein kinase B (AKT) and p-AKT protein expression in immortalized human keratinocyte cell line HaCaT and normal human epidermal keratinocytes (NHEK) after 2 ng/ml transforming growth factor-β1 (TGF-β1) treatment for 0, 6, 12 and 24 h. Transwell assay and Wound healing assay were introduced to measure the cell migration of HaCaT cells. TargetScan online database, luciferase reporter assay and RNA immunoprecipitation (RIP) were employed to confirm the relationship between miR-26a and ITGA5. Results: The RNA expression of miR-26a was down-regulated and ITGA5 protein expression was up-regulated by TGF-β1 treatment in HaCaT and NHEK cells in a time-dependent manner. MiR-26a overexpression inhibited the migration of HaCaT cells induced by TGF-β1 while miR-26a inhibitor enhanced the migration. ITGA5 was a downstream target mRNA and regulated by miR-26a. ITGA5 overexpression reversed the inhibitory effect of miR-26a on migration in HaCaT, while ITGA5 knockdown attenuated the stimulative effect of miR-26a inhibitor in HaCaT via PI3K/AKT signaling pathway. Conclusion: MiR-26a overexpression inhibited TGF-β1 induced HaCaT cells migration via down-regulating ITGA5 through activating the PI3K/AKT signaling pathway.

scholarly journals Curculigoside promotes osteogenic differentiation of ADSCs to prevent ovariectomized-induced osteoporosis

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei-Li You ◽  
Zheng-Long Xu
Keyword(s):  
Bone Loss ◽  
Western Blot ◽  
Osteogenic Differentiation ◽  
Signaling Pathway ◽  
Akt Signaling ◽  
Calcium Deposition ◽  
Phenolic Glycoside ◽  
Akt Signaling Pathway ◽  
Alizarin Red ◽  
Alp Activity

Abstract Background Curculigoside is a natural phenolic glycoside compound produced by Curculigo orchioides Gaertn. This study aimed to explore the effects of curculigoside in promoting the osteogenic differentiation of adipose-derived stem cells (ADSCs) as well as the underlying mechanism. Methods ADSCs were treated with curculigoside at different concentrations (0 μmol/L, 1 μmol/L, 2.5 μmol/L, 5 μmol/L, 10 μmol/L, and 20 μmol/L), and cell viability was assessed by CCK-8 assay. Then, the alkaline phosphatase (ALP) activity was determined, and alizarin red S (ARS) staining was performed to measure the extracellular mineralization of curculigoside. Information about protein-chemical interactions is provided by the search tool for interactions of chemicals (STITCH) database. Then, LY294002 was administered to explore the mechanism by which curculigoside promotes the osteogenic differentiation of ADSCs. Western blot assays were performed to assess changes in the expression of osteogenic-related markers and the phosphorylation of PI3K and AKT. Finally, we established an ovariectomized (OVX)-induced osteoporosis mouse model and administered curculigoside to explore the effects of curculigoside in preventing bone loss in vivo. Results The CCK-8 assay indicated that curculigoside did not induce cytotoxicity at a concentration of 5 μmol/L after 48 h. The ALP and ARS results revealed that the induced group had higher ALP activity and calcium deposition than the control group. Moreover, the curculigoside group exhibited increased biomineralization, ALP activity, and ARS staining compared to the induced and control groups, and these effects were partially inhibited by LY294002. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis indicated that the target genes of curculigoside were mainly involved in the PI3K-Akt signaling pathway. PCR and western blot analysis showed that the expression of RUNX2, ALP, and Osterix was upregulated in curculigoside-treated ADSCs, but this effect was partially reversed by the PI3K inhibitor LY294002. Moreover, the curculigoside-treated group exhibited significantly increased phosphorylation of AKT to P-AKT compared with the osteogenic induction group. After treatment with curculigoside, the mice had a higher bone volume than the OVX mice, suggesting partial protection from cancellous bone loss. In addition, when LY294002 was added, the protective effects of curculigoside could be neutralized. Conclusions Curculigoside could induce the osteogenic differentiation of ADSCs and prevent bone loss in an OVX model through the PI3K/Akt signaling pathway.

scholarly journals PI3K/AKT Signaling Pathway Mediates the Proliferation, Migration and Invasion of Papillary Craniopharyngioma Cells

2020 ◽  
Author(s):  
Lin Zhou ◽  
Cheng Xing Yang ◽  
Lin Chun Fang ◽  
You Yuan Bao ◽  
Zhi Gang Wang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Akt Signaling ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Specific Cell ◽  
Primary Cells ◽  
Akt Signaling Pathway ◽  
Phosphatidylinositol 3 Kinase ◽  
Growth And Survival ◽  
Pi3k Signaling Pathway

Abstract Objective:Craniopharyngiomas are rare, histologically benign but clinically challenging neoplasms. Here, we aimed to interrogate the effect and significance of Phosphatidylinositol-3-kinase (PI3K) signaling pathway on papillary craniopharyngioma (PCP) cell growth and survival.Methods: We used Western blotting (WB) experiments to evaluate the expression of the PI3K/protein kinase B (AKT) in Craniopharyngiomas tissues, relative to health tissues. Primary tumor cells were obtained from fresh PCP samples by cell culture and then determined by cell morphology, immunofluorescence staining and expression of specific cell markers. In this study, PCP cell lines, isolated from fresh PCP samples, were treated with different concentrations of LY294002, a PI3K/AKT signaling inhibitor, to evaluate their proliferation, migration and invasion. We determined the cell proliferation using Cell Counting Kit-8 and colony formation. We then used flow cytometry to evaluate cell apoptosis and cell cycle. In addition, cell migration and invasion levels were determined by wound healing and Transwell assays, respectively.Results: Our data demonstrated that the expression of phosphorylated-PI3K/AKT was upregulated in human craniopharyngioma tissues compared to the normal control tissues. Immunofluorescence assays showed the presence of cytokeratin (pan CK) and vimentin protein (VIM) in the PCP primary cells. Furthermore, inhibition of PI3K/AKT signaling blocks the proliferation, migration and invasion of the PCP primary cells.Conclusions:Taken together, our data robustly demonstrates that the PI3K/AKT signaling pathway mediates the proliferation, migration and invasion of the PCP cells.

scholarly journals Astragaloside positively regulated osteogenic differentiation of pre-osteoblast MC3T3-E1 through PI3K/Akt signaling pathway

2021 ◽  
Vol 16 (1) ◽  
Author(s):  
Wei Bing Jing ◽  
Hongjuan Ji ◽  
Rui Jiang ◽  
Jinlong Wang
Keyword(s):  
Western Blot ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Signaling Pathway ◽  
Real Time Pcr ◽  
Akt Signaling ◽  
Calcium Deposition ◽  
Control Group ◽  
Akt Signaling Pathway ◽  
Western Blot Assay

Abstract Background Osteoporosis is a widespread chronic disease characterized by low bone density. There is currently no gold standard treatment for osteoporosis. The aim of this study was to explore the role and mechanism of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. Methods MC3T3-E1 cells were divided into control and different dose of Astragaloside (10, 20, 40, 50, and 60 μg/ml). Then, ALP and ARS staining were performed to identify the effects of Astragaloside for early and late osteogenic capacity of MC3T3-E1 cells, respectively. Real-time PCR and western blot were performed to assess the ALP, OCN, and OSX expression. PI3K/Akt signaling pathway molecules were then assessed by Western blot. Finally, PI3K inhibitor, LY294002, was implemented to assess the mechanism of Astragaloside in promoting osteogenic differentiation of MC3T3-E1 cells. Results Astragaloside significantly increased the cell viability than the control group. Moreover, Astragaloside enhanced the ALP activity and calcium deposition than the control groups. Compared with the control group, Astragaloside increased the ALP, OCN, and OSX expression in a dose-response manner. Western blot assay further confirmed the real-time PCR results. Astragaloside could significantly increase the p-PI3K and p-Akt expression than the control group. LY294002 partially reversed the promotion effects of Astragaloside on osteogenic differentiation of MC3T3-E1 cells. LY294002 partially reversed the promotion effects of Astragaloside on ALP, OCN, and OSX of MC3T3-E1 cells. Conclusion The present study suggested that Astragaloside promoted osteogenic differentiation of MC3T3-E1 cells through regulating PI3K/Akt signaling pathway.

scholarly journals Yunnan Baiyao Conditioned Medium Promotes the Odonto/Osteogenic Capacity of Stem Cells from Apical Papilla via Nuclear Factor Kappa B Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Xiyao Pang ◽  
Yanqiu Wang ◽  
Jintao Wu ◽  
Zhou Zhou ◽  
Tao Xu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Alkaline Phosphatase ◽  
Osteogenic Differentiation ◽  
Conditioned Medium ◽  
Signaling Pathway ◽  
Cell Counting ◽  
Dependent Manner ◽  
Alizarin Red ◽  
Apical Papilla ◽  
Yunnan Baiyao

Yunnan Baiyao is a traditional Chinese herbal remedy that has long been used for its characteristics of wound healing, bone regeneration, and anti-inflammation. However, the effects of Yunnan Baiyao on the odonto/osteogenic differentiation of stem cells from apical papilla (SCAPs) and the potential mechanisms remain unclear. The aim of this study was to investigate the odonto/osteogenic differentiation effects of Yunnan Baiyao on SCAPs and the underlying mechanisms involved. SCAPs were isolated and cocultured with Yunnan Baiyao conditioned media. The proliferation ability was determined by cell counting kit 8 and flow cytometry. The differentiation capacity and the involvement of NF-κB pathway were investigated by alkaline phosphatase assay, alizarin red staining, immunofluorescence assay, real-time RT-PCR, and western blot analyses. Yunnan Baiyao conditioned medium at the concentration of 50 μg/mL upregulated alkaline phosphatase activity, induced more mineralized nodules, and increased the expression of odonto/osteogenic genes/proteins (e.g., OCN/OCN, OPN/OPN, OSX/OSX, RUNX2/RUNX2, ALP/ALP, COL-I/COL-I, DMP1, DSP/DSPP) of SCAPs. In addition, the expression of cytoplasmic phos-IκBα, phos-P65, and nuclear P65 was significantly increased in Yunnan Baiyao conditioned medium treated SCAPs in a time-dependent manner. Conversely, the differentiation of Yunnan Baiyao conditioned medium treated SCAPs was obviously inhibited when these stem cells were cocultured with the specific NF-κB inhibitor BMS345541. Yunnan Baiyao can promote the odonto/osteogenic differentiation of SCAPs via the NF-κB signaling pathway.

scholarly journals Asiatic Acid Interferes with Invasion and Proliferation of Breast Cancer Cells by Inhibiting WAVE3 Activation through PI3K/AKT Signaling Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-12 ◽  
Author(s):  
Xiao-jun Gou ◽  
Huan-huan Bai ◽  
Li-wei Liu ◽  
Hong-yu Chen ◽  
Qi Shi ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Expression ◽  
Cancer Cells ◽  
Signaling Pathway ◽  
Nude Mice ◽  
Breast Cancer Cells ◽  
Akt Signaling ◽  
Asiatic Acid ◽  
Akt Signaling Pathway ◽  
Xenografted Tumor

Objective. To explore the ability of asiatic acid to interfere with the invasion and proliferation of breast cancer cells by inhibiting WAVE3 expression and activation through the PI3K/AKT signaling pathway. Methods. The MDA-MB-231 cells with strong invasiveness were screened by transwell assay, and plasmids with high expression of WAVE3 were constructed for transfection. The transfection effect and protein expression level of plasmids were verified by PCR and WB. The effects of asiatic acid on cell proliferation and invasion were investigated by flow cytometry. The xenografted tumor models in nude mice were established to study the antitumor activity of asiatic acid. Results. Asiatic acid significantly inhibited the activity of MDA-MB-231 cells, and the expression level of WAVE3 increased significantly in the tissue of ductal carcinoma in situ and was lower than that in the metastasis group. After plasmid transfection, the mRNA and protein expression of WAVE3 increased significantly in the cells. Asiatic acid at different concentrations had an impact on cell apoptosis and invasion and could significantly inhibit the expression of WAVE3, P53, p-PI3K, p-AKT, and other proteins. The T/C(%) of asiatic acid (50 mg/kg) for MDA-MB-231(F10) xenografted tumor in nude mice was 46.33%, with a tumor inhibition rate of 59.55%. Asiatic acid could significantly inhibit the growth of MDA-MB-231 (F10) xenografted tumors in nude mice (p<0.05). Conclusions. Asiatic acid interferes with the ability of breast cancer cells to invade and proliferate by inhibiting WAVE3 expression and activation and the mechanism of action may be related to the PI3K/AKT signaling pathway.

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