mRNA Levels Of Runx2 Research Articles

MicroRNA-23c inhibits articular cartilage damage recovery by regulating MSCs differentiation to chondrocytes via reducing FGF2.

The aim of the study was to explore the role of microRNA-23c in the differentiation of marrow stromal cells (MSCs) to chondrocytes and its potential mechanism. MSCs were first isolated from rat bone marrow for cell culture. Surface antigens of MSCs (CD29 and CD34) were identified by flow cytometry. MSCs were induced for chondrogenic differentiation in MCDM (Mesenchymal Stem Cell Chondrogenic Differentiation Medium) for 0, 3, and 7 days, respectively, followed by detection of RUNX2, microRNA-23c and FGF2 expressions by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Alcian blue staining was performed to access proteoglycan deposition in MSCs transfected with microRNA-23c mimics or inhibitor. Western blot was conducted to detect the protein expressions of ACAN and COL2A1 in MSCs. The binding condition between microRNA-23c and FGF2 was verified by dual-luciferase reporter gene assay. Finally, MSCs were co-transfected with microRNA-23c mimics and FGF2 overexpression plasmid for rescue experiments. On the fourth day of MSCs isolation, MSCs were in an elongated shape. Flow cytometry results showed positive expression of CD29 and negative expression of CD34, which were consistent with MSCs phenotype. QRT-PCR data elucidated that the mRNA levels of RUNX2 and FGF2 gradually increased, whereas microRNA-23c expression decreased with the prolongation of chondrogenic differentiation. Transfection of microRNA-23c mimics in MSCs remarkably elevated microRNA-23c expression. Alcian blue staining showed that microRNA-23c overexpression results in less proteoglycan deposition in MSCs than that of controls. Both mRNA and protein expressions of ACAN and COL2A1 decreased after microRNA-23c overexpression. Dual-luciferase reporter gene assay confirmed that FGF2 binds to microRNA-23c. Further Western blot results demonstrated that FGF2 expression is negatively regulated by microRNA-23c. FGF2 overexpression reversed the inhibitory effects of microRNA-23c on proteoglycan deposition, as well as expressions of ACAN and COL2A1. MicroRNA-23c expression decreases during chondrogenic differentiation of MSCs, which inhibits MSCs differentiation to chondrocytes by inhibiting FGF2.

Baicalin Promotes Osteogenic Differentiation of Human Cementoblast Lineage Cells Via the Wnt/β Catenin Signaling Pathway.

Baicalin constitutes a natural bioactive flavonoid extracted from Scutellaria baicalensis Georgi that mediates bone formation. However, the biological functions of baicalin in cementoblasts remain unclear. The purpose of this study was to examine the effects of baicalin on osteogenic differentiation of human cementoblast (HCEM) cells. HCEM cells were cultured and treated with 0, 0.01, 0.1 or 1 µM baicalin. Alkaline phosphatase (ALP) and runt-related transcription factor 2 (Runx2) mRNA and protein levels were examined by real-time polymerase chain reaction and western blot analysis, respectively. Cell mineralization was assessed using Alizarin red staining. Glycogen synthase kinase-3 beta (GSK3β) phosphorylation was measured in 1 µM baicalin-treated HCEM cells with or without the Wnt signaling pathway inhibitor, DKK-1 using ELISA and western blotting. The protein levels of ALP and Runx2 and the intensity of Alizarin red staining were enhanced by baicalin in a dose-dependent manner compared to that of the non-treated control. The ratio of phosphorylated to total GSK3β increased in the presence of baicalin but was reduced by the addition of DKK-1. Treatment of HCEMs with baicalin up-regulated mRNA levels of ALP and Runx2, which were reduced by DKK-1. In addition, the protein levels of ALP and Runx2, ALP activity, and calcium deposition were also enhanced by baicalin, and these parameters were inhibited by DKK-1. Baicalin enhanced osteogenic differentiation of HCEM cells through the Wnt/beta catenin signaling pathway which may be useful for periodontal tissue regeneration.

Cajanolactone A from Cajanus cajan Promoted Osteoblast Differentiation in Human Bone Marrow Mesenchymal Stem Cells via Stimulating Wnt/LRP5/β-Catenin Signaling.

Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, β-catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/β-catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and β-catenin, inferring that CLA worked by stimulating Wnt/LRP5/β-catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/β-catenin signaling transduction, is a promising anti-osteoporotic drug candidate.

The Influence of Malassez Epithelial Residue in Inflammatory Periodontal Tissues on the Number of Periodontal Stem Cells and Their Differentiation Ability

To investigate the effect of Malassez epithelial surplus in inflammatory periodontal tissues on the number and differentiation ability of periodontal stem cells. 30 patients with periodontitis admitted to the hospital from November 2014 to April 2017 were selected as the observation group. A total of 30 healthy oral examination patients were selected as control group. Periodontal tissue in the 2 groups were taken and the periodontal ligament cells, the complete inflammatory periodontal ligament stem cells by using the low density screening, purification, and periodontal tissue were obtained by using differential digestion method of Malassez epithelial remaining, were observed under inverted microscope in the periodontal tissue Malassez epithelial residual form, will be in the periodontal tissue Malassez epithelial surplus together with inflammatory periodontal ligament stem cells, RT-PCR technique has been applied to the determination of ALP and Runx-2 mRNA expression level to observe the differentiation ability. Flow cytometry showed that the cell markers CD29 and CD90 in the cultured periodontal stem cells were positive (88.9% and 99.7%, respectively). A hematopoietic stem cell marker CD45 was negative (0.3%). The inverted microscope results showed that the residual epithelial cells of Malassez, isolated and cultured, had a large nuclear to cytoplasmic ratio, and the different cells were arranged relatively closely, with the overall cell structure of paving stone. The expression levels of ALP and runx-2 mRNA in the observation group were higher than those in the control group (P<0.05). Malassez epithelial surplus in inflammatory periodontal tissues can promote the proliferation of periodontal stem cells and improve the ability of osteogenic differentiation.

CD137-CD137L interaction induced the calcification of mouse smooth muscle cells via P38 MAPK signaling

Objective: To explore whether CD137-CD137L interaction could induce mouse vascular smooth muscle cells(VSMCs) calcification via P38 MAPK signaling. Methods: (1) Mouse VSMCs obtained from 8-week old male C57 mice were cultured by using method of tissue piece inoculation.The cells from 3 to 8 passage were divided into 4 groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group(agonist-CD137 group+P38 inhibitor), single anti-P38 group(P38 inhibitor). The calcification was induced by adding a mixture of 10 mmol/L β-glycerophosphate+10(-8) mol/L dexamethasone+10(-7) mol/L insulin in the culture medium.Immunofluorescence was used to observe the changes of VSMCs markers(α-SMA and OPN).Real time-PCR was used to observe the mRNA expression of OPN and RUNX-2. Western blot was used to observe the protein expression of p-P38, OPN and RUNX-2. The level of cell calcification was observed by detecting alkaline phosphatase activity and calcium concentration. (2) The degeree of local calcium deposition was also tested on Von Kossa staining and Alizarin red staining methods in following 5 mouse VSMCs groups: control group, agonist-CD137 group(recombinant CD137L protein), anti-P38 group (agonist-CD137 group+P38 inhibitor), anti-CD137 group (agonist-CD137 group+CD137 inhibitor),agonist-P38 group(anti-CD137 group+P38 agonist). Results: (1) Compared with the control group, the fluorescence intensity of α-SMA was lower in the agonist-CD137 group(2.79±0.25 vs. 5.42±0.47,P<0.05), while the fluorescence intensity of OPN was higher(4.91±0.23 vs. 1.63±0.26, P<0.05). The fluorescence intensity of α-SMA was partly recovered after adding P38 inhibitor(4.48±0.27 vs. 2.79±0.25,P<0.05),but it was still lower than the control group (4.48±0.27 vs. 5.42±0.47, P<0.05),the fluorescence intensity of OPN decreased(2.66±0.15 vs. 4.91±0.23,P<0.05),but it was still higher than that in the control group (2.66±0.15 vs. 1.63±0.26,P<0.05).The fluorescence intensity of α-SMA and OPN(5.32±0.67 vs. 5.42±0.47,1.82±0.30 vs.1.63±0.26,both P>0.05) was similar between the control group and single anti-P38 group.(2) Compared with the control group, the protein level of p-P38(4.15±0.24 vs. 3.48±0.26, P<0.05), OPN(2.43±0.21 vs. 1.53±0.08, P<0.05), RUNX-2(3.20±0.23 vs. 1.13±0.10, P<0.05) was significantly increased in agonist-CD137 group,the above effects were blocked by adding specific P38 inhibitor SB203580(1.16±0.12 vs. 4.15±0.24, 0.50±0.02 vs. 2.43±0.21,and 1.74±0.14 vs. 3.20±0.23,all P<0.05);the protein level of p-P38(2.93±0.60 vs. 3.48±0.26,P>0.05),OPN (1.4±0.64 vs. 1.53±0.08,P>0.05),RUNX-2(1.26±0.26 vs.1.13±0.10, P>0.05) was similar between single anti-P38 group and the control group. (3) Compared with the control group, the mRNA level of OPN (1.51±0.34 vs. 1, P<0.05) and RUNX-2(2.67±0.19 vs. 1, P<0.05) was significantly upregulated in agonist-CD137 group, and these effects were blocked by adding specific P38 inhibitor SB203580(0.33±0.14 vs. 1 and 0.45±0.03 vs. 1,P<0.05);the mRNA level of OPN (1.05±0.09 vs. 1, P>0.05) and RUNX-2(1.18±0.10 vs. 1, P>0.05) was similar between the single anti-P38 group and the control group.(4) Compared with the control group,the ALP activity and calcium concentration(2.40±0.25 vs. 1.40±0.21,5.51±0.33 vs. 3.15±0.31,both P<0.05) were significantly increased in agonist-CD137 group,while the effects could be blocked by adding specific P38 inhibitor SB203580((1.99±0.07) king unit/gprot vs. (2.40±0.25) king unit/gprot, (3.74±0.20) mmol/gprot vs. (5.51±0.33) mmol/gprot, both P<0.05).The ALP activity and calcium concentration was similar between single anti-P38 group and the control group((1.60±0.25) king unit/gprot vs. (1.40±0.21)king unit/gprot, (2.66±0.28) mmol/gprot vs. (3.15±0.31) mmol/gprot, both P>0.05). (5) Compared with the control group,the calcification of VSMCs in the agonist-CD137 group was significantly increased,while the calcification in the anti-P38 group was significantly reduced.Compared with the agonist-CD137 group,the level of calcification in the anti-CD137 group was obviously increased,and the calcification in the agonist-P38 group was significantly higher than that in the anti-CD137 group and the control group. Conclusion: These findings suggest that CD137-CD137L signaling may regulate VSMCs calcification via modulating P38 pathway.

MicroRNA-146a reverses the inhibitory effects of Porphyromonas gingivalis lipopolysaccharide on osteogenesis of human periodontal ligament cells

Objective: To investigate the effects and mechanisms of microRNA-146a (miR-146a) on osteogenic differentiation of human periodontal ligament cells (hPDLC) stimulated by lipopolysaccharide (LPS) of Porphyromonas gingivalis (Pg). Methods: hPDLC were cultured in vitro and induced to the phase of osteogenic differentiation. These cells were divided into five groups: non-osteogenic differentiation cells, osteogenic differentiation cells, osteogenic differentiation cells treated with Pg LPS, osteogenic differentiation cells treated with Pg LPS and miR-146a mimic, osteogenic differentiation cells treated with Pg LPS and miR-146a negative control. Osteogenic markers and mineralization were detected via quantitative real-time PCR (qPCR) and alizarin red staining, respectively. Meanwhile, non-radioactive transcription factor assay was applied to explore the nuclear activity of nuclear factor kappa B (NF-κB) p65 in nuclear extracts of hPDLC. Results: Compared with cells of osteogenic differentiation in non-LPS-stimulated groups, Pg LPS could decrease the markers of osteogenic differentiation of hPDLC such as collagen Ⅰ (Col-Ⅰ), alkaline phosphatase (ALP), Runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) (P<0.05), inhibit mineralization, and stimulate NF-κB p65 nuclear activity expression (non-LPS stimulated group: 1.023±0.217, LPS stimulated group: 6.252±0.613, P=0.008). However, compared with cells in Pg LPS/miR-146a negative control group, miR-146a increased Col-Ⅰ (P=0.007) and OCN (P=0.049) mRNA expression, rather than ALP (P=0.167) and RUNX2 (P=0.580) at day 3; miR-146a also upregulated mRNA levels of Col-Ⅰ, ALP, RUNX2 and OCN (P<0.05) at day 7 and day 14, and enhance mineralization. Meanwhile, miR-146a mimic could decrease the nuclear activity of NF-κB p65 induced by Pg LPS in hPDLC (miR-146a: 2.427±0.354, negative control: 5.863±0.482, P=0.019). Conclusions: miR-146a could reverse the inhibitory effects of Pg LPS on osteogenic differentiation of hPDLC through enhancing the expression of osteogenic markers and decreasing inflammatory pathway in hPDLC.

Lentivirus‑mediated microRNA‑26a overexpression in bone mesenchymal stem cells facilitates bone regeneration in bone defects of calvaria in mice

Repair of bone defects presents a serious clinical challenge as it is difficult to restore bone function and regenerate bone loss. In the present study, the effects of lentivirus-mediated transfection of bone marrow mesenchymal stem cells (BMSCs) with microRNA (miR)-26a on bone regeneration were investigated in a mouse bone defect repair model. Marker of proliferation Ki67 (Ki67) staining was employed to detect the cell proliferation capacity and alkaline phosphatase (ALP) staining was used to investigate osteogenic differentiation. A mouse model of cranial bone defects was established. β-tricalcium phosphate biomaterials co-cultured with the transfected BMSCs were implanted into the defect areas of mouse models. Micro-computed tomography, and hematoxylin and eosin and toluidine blue staining, were used to detect bone regeneration in the defect areas and the degradation of scaffolds. miR-26a expression, and the mRNA and protein expression of osteogenesis-associated cytokines, were detected using reverse transcription-quantitative polymerase chain reaction and western blot analysis. Separated and cultured BMSCs highly expressed CD29 and CD105, but not CD34 and CD45, as determined by flow cytometry. miR-26a expression and the positive cell rate of Ki67 and ALP staining in BMSCs transfected with pLVTHM-miR-26a were increased. The BMSC and negative control-transfected BMSC groups exhibited increased bone regeneration in the defect areas, increased bone volume of newly formed bones, and elevated mRNA and protein expression of runt-related transcription factor 2 (Runx2) and osteocalcin (OC), compared with the blank group. However, the miR-26a-transfected BMSC group exhibited further increases in bone regeneration and the volume of newly formed bones, and further elevations of the mRNA and protein expression levels of Runx2 and OC. The present findings demonstrated that lentivirus-mediated modification of BMSCs enhanced bone regeneration during the repair of cranial bone defects in mice.

Serpina3n, Dominantly Expressed in Female Osteoblasts, Suppresses the Phenotypes of Differentiated Osteoblasts in Mice.

It is well known that sex differences exist concerning the severity of osteoporosis and bone metabolism, suggesting that factors other than sex hormones might be responsible for sex differences of bone metabolism. We therefore examined sex differences of osteoblast phenotypes of mouse osteoblasts and then performed comparative gene expression analyses using a comprehensive DNA microarray between female and male osteoblasts. Alkaline phosphatase (ALP) activity, mineralization, and the expression of Osterix, ALP, and bone sialoprotein were significantly lower in mouse female osteoblasts compared with male osteoblasts. We identified Serpina3n, a novel serine protease inhibitor, as the gene whose expression has the highest ratio of females to males. A reduction in endogenous levels of Serpina3n by small interfering RNA significantly enhanced the mRNA levels of Runx2, ALP, osteocalcin, and type I collagen (Col1a1) in both male and female osteoblasts. Moreover, Serpina3n overexpression significantly suppressed the mRNA levels of Osterix, ALP, osteocalcin, and Col1a1 in MC3T3-E1 cells. Serpina3n overexpression did not affect Osterix, ALP, and osteocalcin mRNA levels enhanced by bone morphogenetic protein (BMP)-2 in ST2 cells, adipogenic differentiation in ST2 and 3T3-L1 cells, and receptor activator of nuclear factor κB ligand-induced osteoclast formation in RAW264.7 cells, although it significantly suppressed mineralization in ST2 cells differentiated into osteoblasts by BMP-2. In conclusion, we found Serpina3n as the most female osteoblast-dominant gene. Serpina3n exerts a suppression of the osteoblast phenotypes such as Col1a1 expression and ALP activity in differentiated osteoblasts, which might partly explain sex differences of the osteoblast phenotypes in mice.

Forced expression of mouse progerin attenuates the osteoblast differentiation interrupting β-catenin signal pathway in vitro.

Nuclear protein, lamin A, which is a component of inner membrane on nucleoplasm, plays a role in nuclear formation and cell differentiation. The expression of mutated lamin A, termed progerin, causes a rare genetic aging disorder, Hutchinson-Gilford progeria syndrome, which shows abnormal bone formation with the decrease in a number of osteoblasts and osteocytes. However, exact molecular mechanism how progerin exerts depressive effects on osteogenesis has not been fully understood. Here, we created mouse lamin A dC50 cDNA encoding progerin that lacks 50 amino acid residues at C-terminus, transfected it in mouse preosteoblast-like MC3T3-E1 cells, and examined the changes in osteoblast phenotype. When lamin A dC50-expressed cells were cultured with differentiation-inductive medium, alkaline phosphatase (ALP) activity and mRNA levels of major osteoblast markers, type I collagen (Col1), bone sialoprotein (BSP), dentine matrix protein 1 (DMP1), and Runx2 were significantly decreased, and no mineralized nodules were detected as seen in control cells expressing empty vector. In the culture with mineralization-inductive medium, mRNA levels of BSP, osteocalcin, DMP1, Runx2, and osterix were strongly decreased parallel with loss of mineralization in lamin A dC50-expressed cells, while mineralized nodules appear at 21days in control cells. Furthermore, lamin A dC50 expression was depressed nuclear localization of β-catenin with the decrease of GSK-3β phosphorylation level. These results suggest that lamin A dC50 depresses osteoblast differentiation in both early and late stages, and it negatively regulates β-catenin activity interacting with GSK-3β in cytoplasm.

Bone marrow stem cells-derived exosomes extracted from osteoporosis patients inhibit osteogenesis via microRNA-21/SMAD7.

To explore whether bone marrow stem cells (MSCs)-derived exosomes extracted from osteoporosis patients could inhibit osteogenesis via microRNA-21/SMAD7. MSCs from osteoporosis patients were isolated and cultured. MSCs morphology was observed, and the specific surface antigens were identified by flow cytometry. The osteogenic ability of MSCs was detected by alizarin red staining and oil red staining. Exosomes were collected from MSCs suspension by ultracentrifugation, and microRNA-21 expression in MSCs derived-exosomes was detected. Moreover, protein and mRNA levels of ALP, Bglap, and Runx2 in MSCs treated with different sources of MSCs-derived exosomes were detected by qRT-PCR (quantitative real-time polymerase chain reaction) and Western blot, respectively. ALP activity in MSCs was accessed by a relative commercial kit. Furthermore, binding sites of microRNA-21 and SMAD7 were predicted by Targetscan, miRWalk, and miRDB, and were further verified by luciferase reporter gene assay. SMAD7 expression in MSCs derived-exosomes was also detected. MSCs extracted from healthy adults, and osteoporosis patients were in adherent growth and exhibited elongated morphology, which could differentiate into osteoblasts and lipoblasts after different inductions. MicroRNA-21 expression in MSCs-derived exosomes extracted from osteoporosis patients was remarkably higher than those extracted from healthy adults. Decreased Runx2 expression and ALP activity were found after treatment of MSCs-derived exosomes extracted from osteoporosis patients. SMAD7 was confirmed to bind to microRNA-21 and was downregulated in osteoporosis patients in comparison with healthy adults. Overexpression of SMAD7 resulted in downregulated ALP, Bglap, and Runx2. MicroRNA-21 inhibits osteogenesis through regulating MSCs-derived exosomes extracted from osteoporosis patients via targeting SMAD7.

RUNX2 Promotes Malignant Progression in Glioma.

Glioblastoma (GBM) is the most aggressive and lethal form of brain tumor. However, therapeutic strategies against malignant gliomas have not been completely established. Runt-related transcription factor 2 (Runx2) is an essential gene for skeletal development but its regulatory role in the malignant progression of glioma remains unclear. Here we investigated expression levels of RUNX2 in glioma tissues and its regulatory effects on aberrant growth of glioma cells. RUNX2 mRNA levels were higher in GBM tissues than that of normal brains or low-grade gliomas. RUNX2 protein was detected in five out of seven human GBM cell lines and its level was positively correlated with proliferative capacity. Stable transduction of dominant-negative Runx2 in rat-derived C6 glioma cells not only inhibited the promoter activity containing Runx2 response element, but also decreased mRNA expression levels of Runx2 target genes, such as Mmp13 and Spp1, as well as the proliferative capacity. Furthermore, transient introduction of Runx2-targeted siRNAs into C6 glioma cells significantly decreased mRNA expression levels of Mmp13 and Spp1 and the proliferative capacity. Furthermore, Runx2 knockdown suppressed both Ccnd1 mRNA expression and activation of the Ccnd1 promoter by forskolin, a PKA-activating reagent, in C6 glioma cells. Our results demonstrate that cross-talk between cAMP/PKA signaling and RUNX2 promotes a malignant phenotype of glioma cells.

Localization of leucine-rich repeat-containing G-protein-coupled receptor 5- and Ki67-positive periodontal cells expressing runt-related transcription factor 2 during tooth movement

PurposeMechanical stimuli induce the proliferation and osteogenic differentiation of periodontal ligament (PDL) progenitor cells. However, the localization of formative and proliferative cells in response to orthodontic force in the PDL is unknown. We aimed to elucidate the expression patterns and localization of the cell proliferation- and osteogenesis-associated markers in the PDL during tooth movement. Materials and methodsFive-week-old male Sprague–Dawley rats had their right maxillary first molars (rM1s) moved mesially using a 10-gf titanium-nickel alloy closed-coil spring. Left maxillary first molars (lM1s) served as controls. Immunohistochemical analyses of leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), Ki67, and runt-related transcription factor 2 (RUNX2) were performed in the periapical region of M1. mRNA levels of Ki67, Runx2, and β-catenin were quantified by real-time reverse transcription polymerase chain reaction (qRT-PCR). In vitro human PDL stem cells (hPDLSCs) were cyclically stretched for 24h, and MTT assay and western blot analysis of LGR5 were performed. ResultsImmunohistochemistry showed that LGR5 and Ki67 were partially co-expressed with RUNX2 in the periapical PDL. Immunoreactivities of LGR5, Ki67, and RUNX2 were significantly greater in the PDL of the rM1 than in that of the lM1 on day 3. qRT-PCR showed that Ki67 and Runx2 mRNA levels, but not β-catenin levels, increased on the mesial side of the periapical PDL on day 3. Western blot analysis revealed that 24h of cyclic strain stimulated LGR5 expression in hPDLSCs. ConclusionOur findings suggested that orthodontic force induced LGR-5- and Ki67-positive cells expressing RUNX2 in the PDL during tooth movement.

Wednesday, September 26, 2018 7:35 AM–9:00 AM ePosters

BACKGROUND CONTEXT Alendronate (ALN) is a key therapeutic used to treat glucocorticoid-induced osteoporosis (GIOP), but may induce severe side effects. We showed earlier that plastrum testudinis extracts (PTE) prevented and treated GIOP in vivo. However, clinically, PTE is seldom used alone. PURPOSE We reveal the synergistic effect of ALN and PTE can treat GIOP of the rat spine and define the mechanism. METHODS Sprague-Dawley rats were randomly assigned to four groups: a vehicle group, a GIOP group, an ALN group, and an ALN+PTE group. Each group was further divided into two experimental phases, including dexamethasone (DXM) intervention and withdrawal. Bone mass, microarchitecture, biomechanics, bone-turnover markers and histomorphology were evaluated. The mRNA and protein expression levels of CTSK and Runx2 were detemined. RESULTS We found that ALN+PTE improved bone quantity and quality, bone strength, bone turnover; and mitigated histological damage during glucocorticoid intervention and withdrawal.The therapeutic effect was better than that afforded by ALN alone. ALN+PTE reduced CTSK protein expression, promoted Runx2 mRNA and protein expression to varying extents, and more strongly inhibited bone resorption than did ALN alone. CONCLUSIONS The synergistic effect mediated by ALN+PTE reversed GIOP during DXM intervention and withdrawal via affecting CTSK and Runx2 expression at mRNA and protein levels.

Abstract 4129: RUNX2 as a regulator of high-risk T-ALL

Abstract For patients with T-cell lineage acute lymphoblastic leukemia (T-ALL) who fail induction or relapse, event free survival is &amp;lt;10%. Mixed lineage leukemia gene rearrangements (KMT2A-R) are recurrent translocations found in ~3-5% of T-ALL and are determinants of high-risk disease. However, the roles of downstream targets of KMT2A chimeras such as Runt-related transcription factor 2 (RUNX2) remain uclear. We assessed RUNX2 mRNA levels in 100 pediatric T-ALL samples derived from the COG AALL0434 study. RUNX2 expression was upregulated in patients harboring KMT2A-R (p &amp;lt;0.0005) and those who failed induction therapy (p &amp;lt;0.005). Therefore, we hypothesized that RUNX2 is a critical determinant of high-risk T-ALL. To investigate the relationship between RUNX2 and KMT2A chimeras we utilized the chromatin immunoprecipitation assay to assess primary T-ALL samples with KMT2A-R (n = 3). We found that RUNX2 targets KMT2A-R (p &amp;lt; 0.0005) and conversely, that KMT2A chimeras target the promoter region of RUNX2 (p &amp;lt; 0.0005). We next demonstrated that KMT2A-MLLT4 induces RUNX2 expression. To further test the effects of RUNX2 on KMT2A-R, we performed shRNA mediated silencing of RUNX2 expression in primary T-ALL cells (n =3). RUNX2 silencing decreased KMT2A levels, suggesting the functional cross talk between KMT2A-R and RUNX2. To test the regulatory role of RUNX2 on T-ALL signal transduction we knocked down RUNX2 in T-ALL primary samples and cell lines (Loucy and Jurkat). RUNX2 silencing led to a decrease in cellular proliferation (p &amp;lt; 0.0005) indicating that RUNX2 positively regulates T-ALL cell growth in vitro. We next utilized flow-based assays to examine the effects of RUNX2 on cell death and cell cycle. RUNX2 inactivation induced apoptotic cell death and delayed cell cycle progression as demonstrated by increased G1 and decreased S/G2/M phases of the cell cycle. Apoptotic cell death was associated with increased expression of cleaved caspase 3, 7 and 9. We next examined the effects of RUNX2 on T-ALL cell survival and found that RUNX2 depletion reduced activation of Akt, active β-catenin and survivin levels but had no effect on p53 and Bcl-2. Similar experiments showed that RUNX2 inactivation leads to decreased expression of c-Myc and Ras. In addition, treatment with the specific allosteric Akt inhibitor, MK2206 (10 μM, 24 hrs) reduced RUNX2, active β-catenin, and survivin protein levels suggesting reciprocal activation between RUNX2 and Akt. To study the role of RUNX2 in leukemia burden, Jurkat cells transduced with RUNX2 shRNA and control plasmid were transplanted intravenously into the NSG mice (5 mice/group). RUNX2 silencing significantly decreased leukemia burden in spleen (p &amp;lt;0.001) and bone marrow (p &amp;lt;0.01) of the tested animals compared to the negative control group. We propose upregulation of RUNX2 expression as a novel mechanism governing high-risk T-ALL. We postulate that the Akt/β-catenin/survivin axis serves as a functional driver of RUNX2-mediated T-ALL survival. Citation Format: Nitesh Devinarayan Sharma, Christian K. Nickl, Stuart S. Winter, Huining Kang, Ksenia Matlawska Wasowska. RUNX2 as a regulator of high-risk T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4129.

MiR-383 negatively regulates osteoblastic differentiation of bone marrow mesenchymal stem cells in rats by targeting Satb2.

Emerging evidence indicates that microRNAs (miRNAs, miRs) play diverse roles in the regulation of biological processes, including osteoblastic differentiation. In this study, we found that miR-383 is a critical regulator of osteoblastic differentiation. We showed that miR-383 was downregulated during osteoblastic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Overexpression of miR-383 suppressed osteoblastic differentiation of BMSCs by downregulating alkaline phosphatase (ALP), matrix mineralization, and mRNA and protein levels of RUNX2 and OCN, whereas a knockdown of miR-383 promoted osteoblastic differentiation in vitro. The results of in vivo analysis indicated that inhibition of miR-383 expression enhanced the efficacy of new bone formation in a rat calvarial defect model. Mechanistic experiments revealed that special AT-rich-sequence-binding protein 2 (Satb2) was a direct and functional target of miR-383. Knockdown of Satb2 had inhibitory effects resembling those of miR-383 on the osteoblast differentiation of rat BMSCs. In addition, the positive effect of miR-383 suppression on osteoblastic differentiation was apparently abrogated by Satb2 silencing. Collectively, these results indicate that miR-383 plays an inhibitory role in osteogenic differentiation of rat BMSCs and may act by targeting Satb2.

Heterozygous deletion of LRP5 gene in mice alters profile of immune cells and modulates differentiation of osteoblasts.

Skeletal homeostasis is dynamically influenced by the immune system. Low density lipoprotein receptor-related protein-5 (LRP5) is a co-receptor of the Wnt signaling pathway, which modulates bone metabolism in humans and mice. Immune disorders can lead to abnormal bone metabolism. It is unclear whether and how LRP5 alters the balance of the immune system to modulate bone homeostasis. In this study, we used primary osteoblast to detect the differentiation of osteoblasts in vitro, the immune cells of spleen and bone marrow of 6-month old LRP5 heterozygote (HZ) and wild-type (WT) mice were analyzed by Flow cytometry. We found that LRP5+/- could influence the differentiation of osteoblasts by decreasing the mRNA level of Osterix, and increasing the mRNA level of Runx2 and the ratio of receptor activator for nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG). In the LRP5+/- mice, percentages of NK cells, CD3e+ cells, and CD8a+ T cells were increased in both spleen and bone marrow, and percentages of CD106+ cells and CD11c+ cells were increased in spleen while decreased in bone marrow, conversely, CD62L+ cells were decreased in spleen while increased in bone marrow compared to the WT mice. Percentages of CD4+ cells, CD14+ cells, and CD254+ cells were increased in the spleen, and CTLA4+ cells were increased in the bone marrow of the LRP5+/- mice. The mRNA level of Wnt signaling molecules such as β-catenin, and c-myc were decreased and APC was increased in spleen lymphocytes and bone marrow lymphocytes, and the mRNA level of Wnt3a was decreased in spleen lymphocytes while no change in bone marrow lymphocytes was seen with silencing LRP5 by specific small interfering RNA. In conclusion, heterozygous deletion of the LRP5 gene in mice could alter the profile of the immune cells, influence the balance of immune environment, and modulate bone homeostasis, which might present a potential mechanism to explore the Wnt signaling pathway in the modulation of the immune system.

Effect of bone morphogenetic protein-4 overexpression on the biological activity of mouse induced pluripotent stem cells

This study aimed to construct the expression of bone morphogenetic protein-4 (BMP4) lentiviral vector gene and explore its influence on the biological activity of mouse induced pluripotent stem (iPS) cells. iPS cell lines stably overexpressing BMP4 were constructed by lentivirus transfection (BMP4-overexpressing group). Cells without transfection served as the blank group, and cells with only vector transfection served as the empty-vector group. Cell proliferation was detected by CCK8, and the expression levels of ameloblastin (AMBN), cytokeratin (CK) 14, dentin sialophospho-protein (DSPP), bone sialoprotein (BSP), and Runx2 mRNA were detected by quantitative polymerase chain reaction. Alkaline phosphatase (ALP) activity was used to detect the degree of cell differentiation. Compared with blank and empty-vector groups, proliferation activity and ALP activity of BMP4-overexpressing group obvious increased (P<0.05), BMP4, AMBN, CK14, DSPP, BSP, Runx2 mRNA expression also increased (P<0.05). BMP4 can significantly promote the odontogenic differentiation of iPS.

Therapeutic effect of bone marrow mesenchymal stem cells overexpressing fibroblast growth factor on osteoporotic fracture in rats

Objective To investigate the therapeutic effect of bone marrow mesenchymal stem cells (BMSC) overexpressing basic fibroblast growth factor (bFGF) on osteoporotic fracture in rats. Methods 80 female SD rats were randomly divided into control group, model group, BMSC group and BMSC+ bFGF group. The osteoporotic fracture model was established by resection of ovariectomized in model group, BMSC group and BMSC+ bFGF group. In group BMSC and group BMSC+ bFGF, BMSC and bFGF overexpressing BMSC were transplanted at 1, 14 and 21 d after operation respectively, while rats in the control and model groups were given the same volume of saline injection. The protein level were analyzed by Western blotting in BMSC group and BMSC+ bFGF group. Determination of bone density of rats in four group were detected by bone densitometry and fracture healing was evaluated by X-ray. The changes of bone biomechanical parameters were analyzed using three point bending test. alkaline phosphatase (ALP), runt related transcription factor-2 (Runx2) and bone morphogenetic protein-2 (BMP-2) mRNA level in four groups were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). Results Western blot showed that the expression of bFGF factor was up-regulated in BMSC+ bFGF group. Compared with the model group [2.02±0.37, 0.051±0.002], the X-ray scores and bone mineral density significantly increased in the BMSC [3.12±0.46, 0.800±0.004] and BMSC+ bFGF groups [4.43±0.51, 0.128±0.006], and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.032, 0.013; P=0.000, 0.000). Compared with the BMSC group, the X-ray score and bone mineral density in the BMSC+ bFGF group significantly increased (P=0.010, 0.010) and the biological parameters (maximum load, elastic load, stiffness and maximum deflection) significantly improved (P=0.002, 0.000; P=0.000, 0.000). Compared with the model group (1.41±0.19, 1.31±0.20, 1.43±0.24), the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC group (2.16±0.33, 2.48±0.31, 3.17±0.33) and BMSC+ bFGF group (4.48±0.48, 5.13±0.41, 4.89±0.49) significantly increased (P=0.000). Compared with group BMSC, the levels of ALP, Runx2 and BMP-2 mRNA in callus tissue of BMSC+ bFGF group more significantly increased (P=0.000). Conclusion Overexpression of bFGF in BMSC can increase the expression of bone morphogenetic protein, increase bone mineral density, promote the healing of osteoporotic fracture, and enhance the biological stress of fracture. Key words: Bone marrow mesenchymal stem cells; Basic fibroblast growth factor; Osteoporotic fractures; Bone mineral density; Bone healing; Biology

Cytocompatibility of Biodentine and iRoot FS with human periodontal ligament cells: an invitro study.

To evaluate the cytocompatibility of Biodentine and iRoot FS with human periodontal ligament cells (hPDLCs). Human periodontal ligament cells were characterized by flow cytometry and immunocytochemical analysis. Human periodontal ligament cell adhesion was assessed by scanning electron microscopy at day 3; proliferation by live/dead assay at days 1, 3 and 7; and osteogenic differentiation by alkaline phosphatase (ALP) activity staining, ALP quantification analysis and qRT-PCR at days 7 and 14. Data were analysed with anova and independent sample t-tests with SPSS 21.0. Both iRoot FS and Biodentine increased the adhesion of hPDLCs at day 3. Compared to Biodentine, iRoot FS positively increased hPDLC proliferation on days 3 (P=0.03) and 7 (P=0.00). Osteogenic marker ALP was observed consistently in all samples, with iRoot FS having significantly higher ALP activity at day 14 (P=0.00). Compared with Biodentine, iRoot FS significantly increased the mRNA level of ALP, COL1 and Runx2, and OCN increased only on day 14 (P<0.05). iRoot FS had a positive effect on the adhesion, proliferation and biomineralization of hPDLCs compared with Biodentine.

Carbon monoxide releasing molecule‑3 promotes the osteogenic differentiation of rat bone marrow mesenchymal stem cells by releasing carbon monoxide.

Stem cell‑based therapies are promising strategies to stimulate bone regeneration.Carbon monoxide releasing molecule‑3 (CORM‑3) exhibits multiple regulatory effects in a number of cells by releasing carbon monoxide (CO). The present study aimed to investigate the influence of CORM‑3 on the osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). BMSCs were divided into five groups: A CORM‑3‑osteogenic group, in which cells were pretreated with CORM‑3 and subjected to osteogenic differentiation induction using osteogenic medium; an osteogenic group, in which cells were cultured in osteogenic medium; a degassed CORM‑3‑osteogenic group, in which cells were pretreated with degassed CORM‑3 and subjected to osteogenic differentiation induction; a CORM‑3 group, in which cells were cultured in control medium containing CORM‑3; and a control group, in which cells were cultured in control medium alone. The osteo‑specific mRNA and protein expression of runt‑related transcription factor2 (Runx2), osteocalcin (OCN) and osteopontin (OPN) were assessed using reverse transcription‑quantitative polymerase chain reaction and western blot analysis. Alkaline phosphatase (ALP) activity was also examined and mineralization was detected using alizarin red staining. Levels of Runx2, OCN and OPN mRNA and protein in the CORM‑3‑osteogenic group were significantly increased compared with the osteogenic group (P<0.05), with the exception of OCN protein levels on day3. The mRNA and protein expression of Runx2, OCN and OPN in the degassed CORM‑3‑osteogenic and osteogenic groups were similar. In addition, the mRNA and protein expression of Runx2, OCN and OPN in the CORM‑3 and control group were similar. ALP activity in the CORM‑3‑osteogenic group was increased from day3 and remained significantly higher compared with all other groups on days3,5and7 (P<0.05). Additionally, the results indicated that the optical density value of alizarin red staining in the CORM‑3‑osteogenic group was significantly increased compared with the other groups (P<0.05). Therefore, the present study demonstrated that CORM‑3 may promote the osteogenic differentiation of BMSCs by releasing CO.