| Objective: To investigate the effects of RNAi-mediated down-regulation of human bone morphogenetic protein9(BMP9) on the cross-talkbetween breast cancer cells SK-BR-3and bone marrow stromal cells HS-5inco-culture, and to search for target genes and signal transductionmechanisms may be involved. Then offer experimental basis for the newtherapeutic targets and treatment drugs of breast cancer.Methods: Infected human breast cancer cells SK-BR-3withadenovirus Ad siBMP9, to construct SK-BR-3/AdsiBMP9cells wereendogenous BMP9down-regulated. Then co-cultured indirectly withSK-BR-3cells and human bone marrow stromal cells HS-5with transwellchamber. There were three groups (blank group:SK-BR-3+HS-5,co-Blank;control group: SK-BR-3/Ad RFP+HS-5,co-RFP; experimental group:SK-BR-3/Ad siBMP9+HS-5,co-siBMP9). To detect biological changes ofthe two kinds of cells in co-culture system:for SK-BR-3cells in each group,The capability of proliferation was evaluated by MTT assay; The ability ofsingle cell colony formation was investigated by colony formation test; The ability of apoptosis was investigated by flow cytometry; while themigration capability of SK-BR-3cells and HS-5cells were determined bywound healing test and the transwell migration assay.To detect the mRNA and protein levels changes of related factors inthe two kinds of cells in co-culture system after down-regulating BMP9expression: migration-related factor (SDF-1, IL-6, MCP-1) changes wasdetected by RT-PCR; SDF-1/CXCR4signaling axis as well as HER2expression and activation status was determined by Western blot; and theactivation status of their downstream signaling pathway PI3K/AKT, ERK1/2was evaluated by Western blot; transwell assay without matrigel was used todetermine the migration ability of SK-BR-3cells and Western blot wasapplied to investigate HER2activation status after anti-SDF-1neutralizingantibody was added.The three groups of SK-BR-3cells and HS-5cells were co-culturedindirectly with a1:1proportion for3days, then the mixture weresubcutaneously implanted in nude mice, to construct microenvironmentmodel. The tumor growth in nude mice was observed and recorded on timein each group, the mice were sacrificed after50days and subcutaneoustumors were sectioned. The cells status were observed by HE-stained.migration related indicators were observed by immunohistochemical-stained; Apoptosis was detected by TUNEL-stained.Results: SK-BR-3/AdsiBMP9recombinant cells constructed successfully, and the endogenous BMP9of SK-BR-3/Ad siBMP9cells inco-culture system were significantly reduced at gene and protein levels(P<0.05); while down-regulating BMP9expression could promoteproliferation and single cell colony formation of SK-BR-3cells in co-culturesystem, and inhibited apoptosis (P<0.05); while the wound-healing rate and the number of migrated cells of SK-BR-3cells and HS-5cells were remarkably increased (P<0.05).Related factors of SK-BR-3cells and HS-5cells in co-culture systemwere changed after down-regulating BMP9expression: RT-PCR showedmigration-related factors (IL-6, MCP-1) were significantly increased (P<0.05), and SDF-1was visibly up-regulated in HS-5cells (P<0.05),butthere was no change in SK-BR-3cells (P>0.05), Western blot furtherconfirmed SDF-1remarkably increased at protein level in HS-5cells (P<0.05), and CXCR4protein was found in SK-BR-3cells, but showed nodifference (P>0.05); RT-PCR and Western blot showed HER2, p-HER2were up-regulated (P<0.05); Western blot revealed that the key moleculesp-AKT, p-ERK1/2of SDF-1/CXCR4and HER2downstream signalingpathway was significantly increased (P<0.05); transwell migrationexperiments showed the number of migrated cells of SK-BR-3cells reduced(P<0.05), while Western blot indicated HER2showed little change(P>0.05), but P-HER2significantly reduced (P<0.05) after anti-SDF-1neutralizing antibody was added. Compared with the control group, the growth of the subcutaneoustumor was significantly promoted (P<0.05); HE-stained cells in theexperimental group showed in good status, while the control group occurredmore necrotic cells; immunohistochemical-stained revealed the stainingintensity of Ki67, p-HER2and HER2in experimental group were higher;TUNEL-stained showed the apoptotic index was obviously lower (P<0.05).And the results in vitro were consistent with in vivo.Conclusion: Down-regulating BMP9expression could promote theinteraction between HER2-positive human breast cancer cells SK-BR-3andhuman bone marrow stromal cells HS-5in vitro and in vivo, thus contributedproliferation and inhibited apoptosis of SK-BR-3cells, and promotedmigration of SK-BR-3cells and HS-5cell in co-culture system. Possiblemechanisms were as follows:①activated PI3K/AKT and ERK1/2signalingpathway directly;②activated PI3K/AKT and ERK1/2signaling pathwaythrough up-regulating SDF-1/CXCR4axis and HER2signal, and HER2activation could be inhibited by SDF-1/CXCR4axis blocked. |
PDF Full Text Request