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Background and purpose:Exogenous bone morphogenetic protein 9(BMP9)inhibits the malignant progression of human breast cancer,but its expression is often abnormally low in breast cancer.In this study,we intended to explore the expression and role of epigenetically-modified histone lysine-specific demethylase 4A(KDM4A)in breast cancer,and to investigate the relationship between KDM4A and BMP9 and its possible regulatory mechanism.Methods:The expression of KDM4A in breast cancer and its relationship with BMP9 were analyzed by bioinformatics and verified by real-time fluorescence quantitative polymerase chain reaction(RTFQ-PCR)and Western blot.Chromatin immunoprecipitation(ChIP)verified the regulatory role of KDM4A on BMP9,and RNA stability experiments and CHX protein stability experiments verified the effect of KDM4A in BMP9 expression.Exogenous recombinant MDA-MB-231 cells transfected with KDM4A small interfering RNA(siKDM4A)or infected with siBMP9 adenovirus(Ad-siBMP9)were constructed using RNA interference technology and adenoviruses knocking down BMP9,and the migratory and invasive abilities of the cells were detected by scratch healing assay and transwell assay,respectively.Results:Bioinformatics analysis showed that the expression of KDM4A was significantly higher in breast cancer than in normal tissues,and there was a negative correlation between the expression of KDM4A and that of BMP9 in breast cancer;RTFQ-PCR and Western blot showed that KDM4A was highly expressed in different breast cancer cell lines,and the knockdown of KDM4A significantly up-regulated BMP9.ChIP experiment confirmed that KDM4A could be significantly enriched in the promoter region of BMP9 gene,reducing its histone lysine 36 position instead of position 4 methyl status,thus silencing the expression of BMP9.RNA stability assay and CHX protein stability assay confirmed that KDM4A had no significant effect on the mRNA of BMP9,but could affect its protein degradation.After knocking down KDM4A,the migration and invasion abilities of breast cancer cells MDA-MB-231 were significantly inhibited,and this effect could be partially reversed by knocking down BMP9.Conclusion:KDM4A is highly expressed in breast cancer and breast cancer cell MDA-MB-231,and can silence its expression by down-regulating the level of histone methylation in the promoter region of the BMP9 gene,as well as affecting the stability of BMP9 at the protein level rather than at the level of mRNA,and promoting the migration and invasion of breast cancer.
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Objective To investigate the relationship between serum levels of bone morphogenetic protein(BMP)9,Drosophila mother anti-cerebral palsy protein(SMAD)3 and growth and bone age in children with idiopathic short stature(ISS).Methods A total of 110 children with ISS admitted to the Qingdao Eighth Peo-ple's Hospital from September 2020 to September 2022 were selected as the ISS group,and 110 healthy chil-dren who underwent physical examination in the hospital during the same period were selected as the control group.The serum BMP9 and SMAD3 levels were compared between the two groups.Pearson correlation analysis was used to analyze the correlation between BMP9,SMAD3 and sexual development status,height,weight,body mass index(BMI),osteocalcin(Ost),Leptin,bone age index(BAI),bone age difference(BAD)in children with ISS.Re-ceiver operating characteristic(ROC)curve was used to analyze the diagnostic value of BMP9 and SMAD3 in ISS.Multivariate Logistic regression was used to analyze the risk factors of ISS.Results Compared with the control group,the level of BMP9 was significantly increased and the level of SMAD3 was significantly de-creased in the ISS group(P<0.05).There were significant differences in sexual development status,BMI,BAI,BAD,Ost and Leptin levels between the control group and ISS group(P<0.05).Correlation analysis showed that the serum level of BMP9 was negatively correlated with SMAD3,sexual development status,height,weight,BMI,Ost,Leptin,BAI,and BAD in children with ISS(r=-0.497,-0.523,-0.447,-0.486,-0.501,-0.465,-0.502,-0.434,-0.520,P<0.05).Serum SMAD3 level was positively corre-lated with sexual development status,height,weight,BMI,Ost,Leptin,BAI,and BAD(r=0.432,0.458,0.431,0.465,0.503,0.467,0.515,0.527,P<0.05).ROC curve analysis showed that BMP9,SMAD3 joint in-spection ISS area under curve was higher than the two separate detection(Z=2.774,2.958,P<0.05).Multi-variate Logistic regression analysis showed that serum BMP9 level was an independent risk factor for ISS,and SMAD3 level was an independent protective factor(P<0.05).Conclusion The serum level of BMP93 is in-creased and SMAD3 is decreased in children with ISS,and they are closely related to the growth and bone age of children with ISS,which can be used as molecular markers to evaluate the condition of children with ISS.
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Portopulmonary hypertension (POPH) is an increase in pulmonary artery pressure that occurs on the basis of portal hypertension. As a member of the BMP family, bone morphogenetic protein 9 (BMP9) not only has the osteogenic activity, but can also protect endothelial integrity and maintain vascular homeostasis. This article reviews the pathogenesis of POPH, the physiological expression and role of BMP9, and related research advances in the BMP9 signaling pathway and its involvement in pulmonary hypertension and vascular remodeling, thereby exploring the possibility of BMP9 as a new biomarker for POPH to assist in the diagnosis of POPH.
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Objective To investigate the effect of 1 μmol/L and 10 μmol/L all trans retinoic acid（ATRA） on bone morphogenetic protein 9（BMP9）-induced maturation and differentiation of hepatic progenitor cells. Methods BMP9, BMP9 + 1 μmol/L ATRA and BMP9 + 10 μmol/L ATRA acted on HP14-19, respectively. The expression of albumin-drive gussid（LAB-Glus） was detected by luciferase reporter gene. The mRNA levels of ALB, cytokeratin 18（CK18）, tyrosine aminotransferase（TAT）, apolipoprotein B（ApoB） were detected by Real-time PCR. The expressions of ALB and uridine diphosphate glucuronosyltransferase 1 A（UGT1 A） were detected by immunofluorescence. Periodic acid-schiff（PAS） staining and indocyanine green（ICG） uptake assay were used to detect the metabolism and glycogen synthesis of hepatocytes. Real-time PCR and Western blotting were used to detect the expression of retinoic acid receptor（RAR）α, RARβ、RARγ and BMP9 signal related molecules Samd1, Samd 5 and Samd 8. Ad-siRARα、Ad-siRARβ、Ad-siRARγ infected cells were treated with BMP9+10 μmol/L ATRA, the cell morphology and PAS staining result were observed, the mRNA levels of ALB, CK18, TAT and ApoB were detected by Real-time PCR. Results BMP9 could significantly induce the maturation and differentiation of HP14-19 cells. The morphology of HP14-19 cells looked like polygonal paving stone. The expressions of ALB, CK18, ApoB and UGT1 A were significantly up-regulated. Some cells had the function of metabolic detoxification and glycogen synthesis. Compared with the BMP9 group, BMP9+1 μmol/L ATRA group had more mature morphology and larger volume. The expressions of Alb, CK18, ApoB and UGT1 A were up-regulated significantly（P＜0.05）. The number of ICG and PAS positive cells increased. Compared with the BMP9+1 μmol/L ATRA group, BMP9 + 10 μmol/L ATRA group showed long spindle, spindle and polygonal shapes, and the expression of hepatocyte related markers decreased, and the number of ICG and PAS positive cells decreased. ATRA（1 μmol/L） significantly increased the expression of RARα, RARβ and RARγ. Compared with the 1 μmol/L ATRA group, 10 μmol/L ATRA group only increased the expression of RARα. BMP9 did not affect the expression levels of Samd1, Samd5 and Samd8, but up-regulated their phosphorylation. Ad-siRARα could improve cell morphology and PAS staining induced by 10 μmol/L ATRA, while increased the expression of Alb and CK18（P＜0.05）. Conclusion ATRA（1 μmol/L） can promote the maturation and differentiation of hepatic progenitor cells（HPCs） induced by BMP9, while 10 μmol/L ATRA can weaken the differentiation of hepatic progenitor cells. Excessive ATRA may over activate RARα signal to affect the differentiation of hepatic progenitor cells.
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Objective To investigate whether the low-frequency pulsed electromagnetic fields (LF-PEMFs) can enhance the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) induced by bone morphogenetic protein 9 (BMP9). Methods We isolated CD146＋/STRO-1＋ cells (namely PDLSC) from periodontal ligament cells of healthy human premolars, transfected the PDLSC with BMP9-overexpressing recombinant adenoviruses (Ad-GFP-BMP9), exposed the cells to the different intensities of PEMF stimulation (15 Hz, 0.6，1.2，1.8，2.4，3.0 mT, 1 h/12 h), and then detected the expression of runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), osteopontin (OPN) and osteocalcin (OCN) by qRT-PCR and Western blotting. Results Overexpression of BMP9 significantly promoted the expression of osteogenic markers (Runx2, ALP, OCN and OPN) in PDLSC (P0.05). After the intervention with 1.2, 1.8, 2.4 and 3.0 mT PEMF, the expression levels of osteogenic markers in PDLSC were significantly higher than those exposed to BMP9 alone (P0.05), and reached the peak at 2.4 mT (P0.05), indicating that LF-PEMFs enhanced BMP9-induced osteogenic differentiation of PDLSC, and there was a “window effect”. Conclusion LF-PEMFs stimulation (15 Hz, 1.8 to 3.0 mT) can enhance BMP9-induced osteogenesis of hPDLSCs in vitro.
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Aim To study the relationship between WntlOb and bone morphogenetic protein-9 (BMP9)-induced osteogenic differentiation of mesenchymal stem cells (MSCs), and the molecular mechanisms underlying this process. Methods PCR, Western blot and histochemical staining were used to detect the effect of BMP9 on WntlOb and the effect of WntlOb on BMP9-induced osteogenic differentiation in MSCs. Meanwhile, real-time PCR, Western blot, oil red O staining, and flow cytometry assay were used to analyze the potential mechanism of Wnt10b affecting the function of BMP9. Results Wnt10b could be detected in C3H10T1/2, C2C12, MEFs and MC3T3-E1 cells. BMP9 up-regulated the expression of WntlOb in C3H10T1/2 cells. WntlOb enhanced the capability of BMP9 to increase the level of OCN and mineralization in C3H10T1/2 cells, and silencing Wnt10b attenuated these effects of BMP9. Wnt10b exhibited no substantial effect on cell cycle affected by BMP9, but it enhanced the effect of BMP9 on inducing phosphorylation of Smadl/5/8. While silencing Wnt10b attenuated this effect of BMP9. In addition, Wnt10b inhibited BMP9-induced adipogenic differentiation in C3H10T1/2 cells, and silencing Wnt10b promoted this effect of BMP9. Conclusions Wnt10b can promote BMP9-induced osteogenic differentiation of MSCs, which may be mediated through enhancing BMP/Smad signaling and reducing adipogenic differentiation.
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Aim To investigate the relationship between Wnt11 and the BMP9-induced osteogenic differentiation, and the possible molecular mechanism underlying this process as well. Methods qPCR, histochemical staining and Western blot were used to detect the changes of osteogenic differentiation markers and activities of BMP/Smad and p38 MAPK signal. Luciferase reporter assay was used to detect the activity of BMP/Smad signal. Results BMP9 increased the alkaline phosphatase(ALP) activity, mineralization, expression of osteopontin (OPN) and Wnt11 in C3H10T1/2 cells. Wnt11 was detectable in C3H10T1/2, MEFs, MC3T3-E1 and C2C12 cells. In C3H10T1/2, Wnt11 promoted the effect of BMP9 to increase ALP activity, mineralization, and the expression of Runx-2 and OPN. Wnt11 enhanced the effect of BMP9 on increasing BMP/Smad reporter plasmid transcriptional activity and phosphorylation of Smadl/5/8. Wnt11 also increased the BMP9-induced phosphorylation of p38 MAPK. Inhibition of p38 MAPK attenuated the BMP9-induced ALP activity, mineralization and expression of OPN, but these effects were partially reversed by Wnt11. Conclusions BMP9 can up-regulate Wnt11 in MSCs. Wnt11 can promote the BMP9-induced osteogenic differentiation, which may be mediated by increasing the activity of BMP/Smad and p38 MAPK signaling..
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Objective:To examine the expression of transforming growth factor I(TGF-β1) and bone morphogenetic protein-9 (BMP-9) in human nonunion tissues,and to evaluate the clinical significance.Methods:The number of hypertrophic nonunion tissue samples and atrophic nonunion tissue samples were collected from Department of Orthopedics,the Second Xiangya Hospital of Central South University and Suzhou Kowloon Hospital Affiliated to School of Medicine of Shanghai Jiao Tong University between 2010 and 2014.Semi-quantification of SP immunohistochemical method and pathological image analysis software IPP6.0 were used to analyze the expression of TGF-β1 and BMP-9.Nonunion type,patients' age and nonunion time were statistical analyzed.Results:The absorbance values of TGF-β1 and BMP-9 in the hypertrophic nonunion tissues were 0.3236±0.0390 and 0.1337±0.0400,respectively;while the absorbance values of TGF-β1 and BMP-9 in the atrophic nonunion tissues were 0.3191±0.0369 and 0.1373±0.0423,respectively,with no significant difference between the two types of tissues (both P＞0.05).There was also no significant difference in patients' age and bone nonunion time between them (all P＞0.05).Conclusion:There is no significant difference in osteogenic potential between the hypertrophic nonunion tissues and the atrophic nonunion tissues.
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Objective The aim of this study was to investigate the effects of the ERK5 pathway on bone morphogenetic protein?9(BMP9)?regu?lated osteogenic/odontogenic differentiation of immortalized stern cells from the apical papilla(iSCAP). Methods BMP9 was introduced into the iSCAP by using recombinant adenoviruses,and the P?ERK5 protein expression was measured via western blotting. Then,the osteogenic/odontoblas?tic changes were analyzed by alkaline phosphatase(ALP)staining and Alizarin red staining,and the expression of osteogenic/odontoblast?associat?ed genes,such as Runx2,OCN,OPN,and DMP1 was measured by RT?PCR. Results BMP9 could up?regulate the phosphorylation of ERK5 in iSCAP. After using BIX02189,which was an inhibitor of ERK5,the phosphorylation of ERK5;the activity of ALP;the expression of Runx2, OCN,OPN,and DMP1;and the calcium deposition were all significantly inhibited. Conclusion The ERK5 signaling pathway plays an important role in BMP9?regulated osteogenic/odontogenic differentiation of iSCAP.
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Objective:To investigate the effects of BMP9 combined with NGF on the osteogenic differentiation of mesenchymal stem cells(MSCs).Methods:Recombinant BMP9 adenovirus was transfected into C3H10T1/2 cells.The cells were treated by GFP,NGF,BMP9 and BMP9 + NGF respectively.The expression level of COL1,RUNX2 was detected by RT-PCR and Western blot,ALP activity was examined by ALP kit 3,12,24,48 hours,3 and 7 days after treatment,respectively.Results:The ALP activity of BMP9 + NGF group was the highest among the 4 groups.The difference in the groups firstly appeared at 3 h after treatment.The highest expression level of RUNX2 and COL1 was detected in BMP9 + NGF group.Conclusion:NGF and BMP9 may synergisticly promote osteogenic differentiation at the early stage of osteogenic induction of C3H10T1/2 cells.
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Aim To investigated the possible effect of COX-2 on the BMP9-induced activation of PI3K/Akt signal in progenitor cells.Methods The activity of alkaline phosphatase(ALP) was measured using histochemical staining or chemiluminescence.The mRNA level of ALP was determined using real-time PCR assay.The protein levels of osteopontin(OPN), osteocalcin(OCN), COX-2, Akt1/2 and phosphorylated Akt1/2 were detected by Western blot.The mRNA level of COX-2 was assayed with RT-PCR, and the mineralization was measured with Alizarin Red staining.Results The ALP activity was apparently increased by BMP9 in C2C12 cells, as well as the protein level of OPN and OCN.The mineralization was also markedly induced by BMP9 in C2C12 cells.BMP9 increased the level of phosphorylated Akt1/2 greatly, although no substantial effect was observed on total protein level of Akt1/2.The BMP9-induced ALP activity was dramatically decreased by the inhibitor of PI3K.The mRNA and protein level of COX-2 were both increased by BMP9 in C2C12cells, and the BMP9-induced ALP activity and mineralization were greatly attenuated by the inhibitor of COX-2.The BMP9-induced phosphorylation level of Akt1/2 was increased by the exogenous expression of COX-2, but decreased by the inhibitor of COX-2.Conclusion Activation of PI3K/Akt signaling may be a critical event in BMP9-induced osteogenic differentiation, and this process may be mediated by the BMP9-upregulated COX-2 in stem cells at least.
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AIM:To investigate the effect of bone morphogenetic proteins 9 (BMP9) on the migration and in-vasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism.METHODS:The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial ( HBE) cells was detected by RT-PCR and Western blot.The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expres-sion of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot.The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays.The mRNA and protein levels of the migra-tion-related factor matrix metalloproteinase 2 (MMP2) were detected by RT-PCR and Western blot.The level of phospho-rylated Smad1/5 (p-Smad1/5) was detected by Western blot.Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9.The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays.RESULTS:The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells.After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly de-creased, and the mRNA and protein levels of MMP2 were decreased.Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability.CONCLUSION:O-ver-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells.The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.
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Objective: To investigate the effect of β-catenin and bone morphogenetic protein 9 (BMP-9) on osteogenic differentiation of osteosarcoma TE85 cells. Methods: The TE85 cells were infected by recombinant adenovirus Adβ-catenin and AdBMP9 alone or in combination. The expression levels of β-catenin and BMP9 mRNAs in TE85 cells were detected by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). The relative β-catenin/TCF4 activity was detected by Luciferase Reported System. Alkaline phosphatase (ALP) staining and numeration were used to detect the early osteogenic potential of TE85 cells, and the alizarin red staining was performed to detect the late osteogenic potential. The expression levels of osteoprotegerin (OPG), osteocalcin (OC) and osteopontin (OPN) mRNAs were detected by semi-quantitative RT-PCR, and the expression levels of OC and OPN proteins were detected by Western blotting. Results: The exogenous mRNAs of β-catenin and BMP9 in TE85 cells were increased by adenovirus-mediated Adβ-catenin and AdBMP9 (both P < 0.05), and the relative β-catenin/TCF4 activity was increased after infection with Adβ-catenin (P < 0.05). Adβ-catenin or AdBMP9 infection could enhance the activity of ALP in a limited range in TE85 cells (P < 0.05), but they could not promote the mRNA and protein expressions of late osteogenic biomarkers OPN and OC as well as the calcium deposition. However, both early and late potential osteogenic abilities were enhanced obviously after infection with combination of Adβ-catenin and AdBMP9 (all P < 0.05). Conclusion: Osteogenic differentiation can be limitedly induced by β-catenin and BMP9 alone in TE85 cells, but this differentiation ability can be remarkably enhanced by combination of β-catenin and BMP9 with a synergistic effect.
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Objective: To investigate the effects of bone morphogenetic protein 9 (BMP9) on the proliferation and apoptosis of breast cancer MDA-MB-231 cells in vitro and in vivo. Methods: MD-AMB-231 cells were infected with pAdtrack-CMV/BMP9 or pAdtrack-CMV/GFP adenovirus. The expression levels of BMP9 mRNA in MDA-MB-231/GFP and MDA-MB-231/BMP9 cells were detected by RT-PCR. The proliferation inhibitory rate and the apoptosis rate of breast cancer cells were determined by MTT assay, colony-forming assay and flow cytometry method. Then the breast cancer MDA-MB-231, MDA-MB-231/GFP or MDA-MB-231/BMP9 xenografts in nude mice were established. The volume of xenograft tumor was measured, and the apoptosis rate was detected by TUNEL assay. Results: There was no expression of BMP9 mRNA in MDA-MB-231 and MDA-MB-231/GFP cells, but the expression of BMP9 mRNA in MDA-MB-231/BMP9 cells was significant. The proliferation inhibitory rate of MDA-MB-231/BMP9 cells was higher than that of MDA-MB-231/GFP cells ( P<0.05). The colony-forming rate of MDA-MB-231/BMP9 cells was lower than those of MDA-MB-231/GFP and MDA-MB-231 cells ( P<0.05). The apoptosis rate of MDA-MB-231/BMP9 cells was higher than those of MDA-MB-231/GFP and MDA-MB-231 cells ( P<0.05). The xenograft tumor volume in the MDA-MB-231/BMP9 group was smaller than those in the MDA-MB-231/GFP and MDA-MB-231 groups ( P<0.001). The apoptosis index of xenograft tumor in the MDA-MB-231/BMP9 group was higher than those in the MDA-MB-231/GFP and MDA-MB-231 groups ( P<0.001). Conclusion: BMP9 can inhibit the proliferation ability and induce the apoptosis of MDA-MB-231 cells in vitro and in vivo.
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In order to validate and estimate the capability of BMP9 to induce osteogenic differentiation of multipotent stem cells, three multipotent stem cells(C3H10, MEFs and BMSC) were used as target cells, and BMP9 was introduced into these cells by using recombinant adenoviruses assay, the effect of BMP9 on osteogenic differentiation of multipotent stem cells was demonstrated by using luciferase reporter assay, alkaline phosphatase(ALP) quantitative assay, calcium deposition assay, real time PCR, animal experiment and histological staining assay.The results demonstrated that BMP9 can induce ALP expression of C3H10, MEFs and BMSC by a dose dependent manner.BMP9 can also stimulate calcium deposition of C3H10 and MEFs in vitro, the osteogenic markers(ALP, Runx2, osteopontin, osteocalcin) were increased after stimulated by BMP9.BMP9 can activate canonical TGF?-Smad pathway, and promote the expression of osteogenic master gene Runx2.The animal experiment and histological staining assay show that BMP9 can induce ectopic bone formation in naked mice.To sum up, BMP9 is a more powerful cell factor to induce osteogenic differentiation of multipotent stem cells.