| Objective:Transforming growth factor-β (TGF-β) is known to promote tumormigration and invasion. Bone morphogenetic proteins (BMPs) are membersof the TGF-β family expressed in a variety of human carcinoma cell lines.Bone morphogenetic protein9(BMP9), the most powerful osteogenic factor,in our previous study, we found that BMP9overexpression might inhibitsOsteosarcoma (OS) cell growth and migration, however, the role of BMP9in OS progression has not been fully clarified. Then, in this study, we aimedto define the role of BMP9in OS cells and explore its molecularmechanisms.Methods:The expression of BMP2,4,6,7,9and type I [activin receptor-likekinase (ALK1-7)] and type II (BMPRII, ActRIIA and ActRIIB) BMPreceptors in OS cell lines143B and MG63was analyzed by RT-PCR. Theefficiency of AdBMP9and AdsiBMP9in OS cell lines143B and MG63was confirmed by RT-PCR. High titer of adenovirus was generated byHEK293cells, and then infected OS cell lines143B and MG63. Experimental group: AdBMP9, AdGFP, AdsiBMP9, AdRFP, Blank. Cellviability was examined by MTT assay, cell cycle distribution and apoptosiswere analyzed by flow cytometry. Cell motility and invasion were analyzedby wound healing assay and transwell migration and invasion assay.The expression of BMP/Smads related molecules was analyzed byRT-PCR and western blot analysis. The transcription activity of BMP9onBMPR-Smad luciferase reporter plasmid p12SBE-luc promoters wasassayed by luciferase reporter gene assay kit.The expression of wnt/β-catenin related molecules was analyzed byRT-PCR and western blot analysis. Preparation of conditioned mediumBMP9,143B cells were treated with Adβ-catenin, Adsiβ-catenin or thecontrol adenovirus AdGFP, AdRFP. After infecting for8-12h, themedium was replaced with BMP9conditional medium. Cell viability wasexamined by MTT assay, cell migration was analyzed by transwellmigration assay. The expression of β-catenin downstream target genesc-myc and OPG were analyzed by western blot.The effect of BMP9on CXCL12/CXCR4axis was detected byRT-PCR, ICC and ELISA. The expression of MMP2, MMP7and MMP9was detected by RT-PCR and qRT-PCR. The activity of MMP2and MMP9was determined by gelatin zymography.Results:In both143B and MG63cells, BMP2,4，6，7，9and all BMP receptors were readily detected，and among BMPs, the expression of BMP9was thelowest. BMP9overexpression significantly inhibited the cell viability ofOS cells at24h and tended to be more significant at48and72h, whereasBMP9silencing significantly promoted the cell viability of OS cells only at48h (P <0.05). BMP9overexpression decreased the wound closure rateand migrating cell numbers from170±19.1to98±12.9(P <0.05) in143B cells and64±7to40±4.5(P <0.05) in MG63cells compared withthe AdGFP group, BMP9overexpression significantly reduced invasioncell numbers from16.3±0.9to9±1.8(P <0.05) in143B cells and from13±1.2to6.3±0.8(P <0.05) in MG63cells compared with the AdGFPgroup, whereas BMP9silencing had opposite effects. BMP9overexpression decreased the percentage of143B and MG63cells in the Sphase from (59.32±5.16)%to (39.07±4.16)%(P <0.05) and (42.75±4.69)%to (9.41±3.82)%(P <0.05) compared to the AdGFP group, andincreased the percentage of143B and MG63cells at the G1phase of thecell cycle from (31.14±3.29)%to (47.83±4.66)%(P <0.05) and (43.36±3.11)%to (82.65±3.68)%(P <0.05) compared to the AdGFP group,respectively. The apoptosis rate of143B cells was significantly increasedfrom (10.23±2.61)%to (19.91±2.29)%(P <0.05) compared to AdGFPgroups after BMP9treatment for48h, whereas the apoptosis rate of MG63cells was not significantly higher than that of the AdGFP groups afterBMP9treatment for48h (P>0.05). BMP9overexpression increased the phosphorylation of Smad1/5/8proteins, and that BMP9knockdown reduced the phosphorylation ofSmad1/5/8proteins (P <0.05), while total Smad1/5/8proteins did notchange, in143B and MG63cells.2.9-fold and2.1-fold increases inpromoter activity were observed in143B and MG63cells, respectively,treated with AdBMP9compared with cells treated with AdGFP (P <0.05).BMP9overexpression significantly increased ID1mRNA expression,whereas knockdown of BMP9reduced ID1mRNA expression (P <0.05).The expression of β-catenin and its target genes c-myc and OPG weresignificantly reduced at both the mRNA and protein levels in OS cells afterinfection with AdBMP9for24h, whereas they reverted after infection withAdsiBMP9. BMP9overexpression significantly reduced GSK-3βSer9phosphorylation in OS cells and the suppressive effect was reverted byinhibition of BMP9. The proliferation and migration potency of exogenousβ-catenin expression of143B cells treated with BMP9was restoredcompared with AdGFP infection treated with BMP9, whereas knockout ofβ-catenin of143B cells treated with BMP9was further enhanced comparedwith AdRFP infection treated with BMP9. β-catenin target genes c-myc andOPG expression were restored after exogenous β-catenin expression of143Bcells treated with BMP9, whereas they were further reduced after knockoutof β-catenin expression of143B cells treated with BMP9, compared withAdGFP or AdRFP infection treated with BMP9, respectively. The expression of BMP9did not affect the CXCL12/CXCR4axis.Interestingly, CXCL12was detected in MG63cells but not in143B cells,while CXCR4was expressed in both OS cell lines. BMP9overexpressionmarkedly reduced the levels of MMP9mRNA (P <0.05), whereas BMP9silencing increased the levels of MMP9mRNA (P>0.05). However, theexpression of MMP2and MMP7mRNAs was not regulated by BMP9(P>0.05). BMP9overexpression decreased both the pro-and active forms ofMMP9(P <0.05), whereas BMP9silencing had the opposite effect. MMP2activity was very weak and did not show any change in response totreatment in143B cells.Conclusions:Our results revealed that BMP9overexpression can inhibits the growth,migration and invasion of OS cells. BMP9can regulate tumor growth andmigration of OS cells might through Smad-dependent pathway andWnt/β-catenin pathway and downregulating the mRNA level and activity ofMMP9. BMP9may not affect CXCL12/CXCR4axis to regulate theinvasiveness and metastatic capacity of OS cells. |
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