The Effects And Underlying Mechanism Of BMP9on Proliferation And Migration Of Breast Cancer SK-BR-3Cell Line

Objective To explore the effects of BMP9on the proliferation andmigration of breast cancer SK-BR-3cells in vitro and vivo. To search thepotential downstream target gene and underlying signaling pathway ofBMP9in SK-BR-3cells and offer experimental data for the therapeuticagent of breast cancer.Methods We chose the breast cancer SK-BR-3cell line as the experimentalmodel and we try to elucidate the effects of BMP9on SK-BR-3cells fromgene silencing and gene overexpressing aspects.①We constructed BMP9interfering adenoviruses (AdsiBMP9) and negative control adenoviruses(AdsiNC). The SK-BR-3cells were infected with the AdsiBMP9andAdsiNC adenoviruses to prepare the SK-BR-3/siBMP9cells as theinterfering BMP9group, SK-BR-3/siNC cells as the negative control group,SK-BR-3cells as the blank group;②The SK-BR-3cells were infectedwith the BMP9expression adenoviruses and green fluorescence proteincontrol adenoviruses which were offered by molecular medicine laboratory,to prepare the SK-BR-3/BMP9cells as the BMP9overexpression group, SK-BR-3/GFP cells as the vehical group, SK-BR-3cells as the blank group.MTT assay was used to investigate the ability of growth in differentgroups; Colony-forming assay was applied to evaluate the ability ofcolony-forming in different groups; Wound healing assay and transwellassay without matrigel were hired to investigate the ability of migaration indifferent goups. The expression of HER2in different groups wasinvestigated by RT-PCR and Western blot, the status of MAPK andPI3K/AKT signaling pathways were examined by Western blot.Nude mice xenograft models were constructed to confirm the effectsof BMP9in vivo. The SK-BR-3/siBMP9cells, SK-BR-3cells,SK-BR-3/BMP9cells were injected subcutaneously into nude micerespectively.The diameter of the tumors were recorded every five days. Thenude mice were sacrificed and tumors were obtained after55days，thetumor tissues were collected and fixed in buffered formaldehyde,embedded in paraffin and sectioned for further histological andimmunohistochemical analysis.Result We successfully constructed BMP9interfering adenoviruses（AdsiBMP9） and the negative control adenoviruses(AdsiNC). Theinhibitory rate of AdsiBMP9was58.27%and51.63%from mRNA andprotein levels respectively.Firstly, we investigated the effect of BMP9on proliferation andmigration of SK-BR-3cells and the underlying mechanism.①The result of MTT showed that on the fourth day, the SK-BR-3/siBMP9cellsproliferation rate（0.851±0.0314）were significantly higher than those ofSK-BR-3/siNC cells (0.721±0.0289) and SK-BR-3cells (0.742±0.026)(P＜0.05);②The result of colony-forming assay showed that, on thefourteen day the colony number of SK-BR-3/siBMP9cells（143±5.2）wasdramatically higer than those of SK-BR-3/siNC （88±4.8） andSK-BR-3cells (93±5.1）(P＜0.05)；③The result of wound healingshowed that the healing rate of SK-BR-3/siBMP9cells (0.563±0.032)weremarkedly higher than those of the SK-BR-3/siNC cells（0.563±0.032）andSK-BR-3cells (0.578±0.04) cells(P＜0.05);④The result of transwellwithout matrigel showed that the number on the low chamber ofSK-BR-3/siBMP9cells (146±5.3）were significantly higer than those ofSK-BR-3/siNC cells (89±4.2）and SK-BR-3cells (87±4.5)(P＜0.05)。By RT-PCR we found that the expression of HER2inSK-BR-3/siBMP9cells were (1.381±0.056）and（1.27±0.052）folds thanthose in SK-BR-3/siNC cells and SK-BR-3cells respectively (P＜0.05).By Western blot we found that the protein expressions of p-HER2inSK-BR-3/siBMP9cells were （1.77±0.065）and（1.65±0.058）folds (P＜0.05) than those in SK-BR-3/siNC and SK-BR-3cells respectively, at thesame time the protein expressions of HER2in SK-BR-3/siBMP9cells were(1.42±0.064) and (1.38±0.059） flolds (P＜0.05) than those inSK-BR-3/siNC and SK-BR-3cells respectively. By Western blot we found that the p-ERK1/2protein expressions inSK-BR-3/siBMP9cells were (1.46±0.07) and (1.41±0.04) folds(P＜0.05)than those in SK-BR-3/siNC cells and SK-BR-3cells; The p-AKT proteinexpressions in SK-BR-3/siBMP9cells were (1.52±0.074) and(1.56±0.068) folds(P＜0.05) than those in SK-BR-3/siNC cells andSK-BR-3cells.The results of nude mice xenograft model showed that the nude micewith SK-BR-3/siBMP9cells formed the tumor on the sixteenth day whilethe nude mice with SK-BR-3cells on the nineteenth day; On thefifty-fifth day, the tumor volume of SK-BR-3/siBMP9mice was（1149±210）mm3and appeared serious fester, while the tumor volumeof SK-BR-3mice was（268±136）mm3and the fester was not so serious;The mice were sacrificed and the tissues were fixed in formaldehyde,embedded in paraffin and sectioned, the results of immunohistochemicalanalysis showed that the staining extent of p-HER2、HER2、p-ERK1/2、p-AKT in SK-BR-3/siBMP9mice were stronger than those in SK-BR-3mice.To further explore the effect of BMP9on SK-BR-3cells, weconstructed BMP9overexpression SK-BR-3/BMP9cells, investigated theeffect and underlying mechanisem of BMP9on SK-BR-3cells proliferationand migration.①The result of MTT showed that on the fourth day, theproliferation rate of SK-BR-3/BMP9cells (0.554±0.032) were significantly lower than those of SK-BR-3/GFP cells (0.712±0.033) andSK-BR-3(0.742±0.026) cells (P＜0.05);②The result of colony-formingassay showed that, on the fourteen day the colony number ofSK-BR-3/BMP9cells (47±3.8) was dramatically lower than those ofSK-BR-3/GFP(81±4.4) and SK-BR-3cells (93±5.1）(P＜0.05)；③Theresult of wound healing showed that the healing rate of SK-BR-3/BMP9cells (0.42±0.033) were markerdly lower than those of SK-BR-3/GFPcells (0.621±0.043) and SK-BR-3cells (0.578±0.04) cells(P＜0.05);④The result of transwell without matrigel showed that the number on the lowchamber of SK-BR-3/BMP9cells (54±3.4）were significantly lower thanthose of SK-BR-3/GFP cells (92±6.2）and SK-BR-3cells (87±4.5)(P＜0.05)。We found that the expression of HER2in SK-BR-3/BMP9cells were(0.571±0.036）and（0.543±0.038）folds than those in SK-BR-3/GFP cellsand SK-BR-3cells(P＜0.05) respectively by RT-PCR.By Western blot we found that the protein expressions of p-HER2inSK-BR-3/BMP9cells were（0.545±0.043）and（0.521±0.037）folds (P＜0.05) than those in SK-BR-3/GFP and SK-BR-3cells respectively, at thesame time the protein expressions of HER2in SK-BR-3/BMP9cells were(0.481±0.027) and (0.511±0.043） flolds (P＜0.05) than those inSK-BR-3/GFP and SK-BR-3cells respectively.we found that the p-ERK1/2protein expressions in SK-BR-3/BMP9 cells were (0.423±0.034) and (0.457±0.045) folds(P＜0.05) than those inSK-BR-3/GFP cells and SK-BR-3cells; The p-AKT protein expressions inSK-BR-3/BMP9cells were (0.381±0.028) and (0.402±0.031) folds(P＜0.05) than those in SK-BR-3/GFP cells and SK-BR-3cells.The results of nude mice xenograft model showed that the nude micewith SK-BR-3/BMP9cells formated the tumor on the twenty-third daywhile the nude mice with SK-BR-3cells on the nineteenth day; On thefifty-fifth day, the tumor volume of SK-BR-3/BMP9mice was（65±6.7）mm3and without fester, while the tumor volume of SK-BR-3mice was（268±136）mm3and with slightly fester; The mice were sacrificed andthe tissues were fixed in formaldehyde, embedded in paraffin andsectioned, the results of immunohistochemical analysis showed that thestaining extent of p-HER2、HER2、p-ERK1/2、p-AKT in SK-BR-3/BMP9mice were weaker than those in SK-BR-3mice.Conclusion BMP9could inhibit the proliferation and migration of breastcancer SK-BR-3cells in vitro and in vivo through down-regulating thephosphorylation level of MAPK and PI3K/AKT pathway directly andindirectly by inhibiting the expression of HER2.