Expression Of BMP7 In Ovarian Cancer And Its Effect On Biological Behavior Of Ovarian Cancer Cell

BackgroundOvarian cancer(OC)is a common gynecological malignancy,and its mortality is the highest among gynecological malignancies.Bone morphogenetic proteins(BMPs),among the members of the transforming growth factor-P superfamily,are widely involved in regulating the biological processes of proliferation,differentiation and apoptosis of a variety of cells.BMP7 can bind to receptors on the cell membrane,and then Smad 1/5/9,which is the receptor-regulated Smad(R-Smad),is phosphorylated and activated,regulating multiple target genes.In a previous study,it was reported that the BMP7 gene was overexpressed in chemotherapy-resistant ovarian cancer tissues or cells.Nevertheless,the expression of BMP7 in ovarian cancer and normal ovarian tissues and the biological effect of BMP7 on OC cell remain unclear.In this study,we explored that the expression level of BMP7 in ovarian cancer tissue and normal ovarian tissue,and that the influence of BMP7 knockdown on the cell proliferation,cell migration and invasion,cell cycle distribution and Taxol sensitivity of ovarian cancer cell,as well as changes in the expression levels of R-Smad and ID2.Materials and MethodsSamples from 55 patients with human ovarian cancer and 40 from patients with normal ovarian tissue were collected,and immunohistochemistry was used to detect the expression level of BMP7 protein in ovarian cancer and normal ovarian tissues.The expression level of BMP7 in the A2780 cells was down-regulated by lentivirus vector containing the knocked down BMP7 gene which was transfected into A2780 cell.The cells were divided into the negative control(sh-NC)and the BMP7 down-regulated group(sh-BMP7).The proportion of cells expressing green fluorescent protein(GFP)was observed by fluorescence microscopy,and the mRNA levels of BMP7 and ID2 in sh-BMP7 group were detected by RT-qPCR.Western blotting was performed to detect the expression levels of Smad 1/5/9,p-Smad 1/5/9,ID2,cyclinDl and EMT-related protein(E-cadherin,N-cadherin,Vimentin,Slug and Snail)in A2780 cells with down-regulated BMP7.The protein expression levels of BMP7 and p-Smad 1/5/9 in sh-BMP7 group were detected by cell immunofluorescence.CCK-8 proliferation and clone formation assays were used to observe the effect of down-regulated BMP7 on the proliferation of A2780 cells.Flow cytometry was used to detect the effect of down-regulated BMP7 on the cell cycle.CCK-8 toxicity test and flow cytometry were used to detect the effect of down-regulated BMP7 on the sensitivity of A2780 cells to paclitaxel.Results1.Expression of BMP7 in the ovarian cancer and normal ovarian tissuesThe staining of BMP7 was mainly concentrated in the cytoplasm and stroma of the tissues.Positive expression rate of BMP7 was statistically higher in the ovarian cancer samples than in the normal ovarian specimens(P<0.05).2.Detection of lentivirus transfection efficiencyThe lentivirus transfected cell could express green fluorescence protein.By fluorescence microscopy,the percentage of cell expressing green fluorescent protein was more than 95%.Compared with sh-NC group,the BMP7 mRNA level was decreased in sh-BMP7 group by RT-qPCR(P<0.05).The result of western blotting showed that the expression level of BMP7 protein in sh-BMP7 group was decreased compared with that in sh-NC group.Immunofluorescence result showed that the expression level of BMP7 protein in sh-BMP7 group was decreased.3.Down-regulated BMP7 inhibited ovarian cancer cell proliferationThe CCK-8 proliferation assay revealed that the sh-BMP7 group exhibited a significant decrease in proliferation rate compared with that of the sh-NC group(P<0.05).The result of clone formation experiment showed that the number of cell clones formed in sh-BMP7 group was lower than that in sh-NC group(P<0.05).4.Down-regulation of BMP7 resulted in cell cycle arrest in ovarian cancer cellThe cell proportion of sh-BMP7 group in the G1 phase was significantly increased compared with that of the sh-NC group(P<0.05),while the proportion of cell in the S phase in the sh-BMP7 group was significantly decreased compared with that of the sh-NC group(P<0.05).In addition,the expression level of cyclin D1 was significantly decreased in the sh-BMP7 group compared with that of the sh-NC group.5.Down-regulation of BMP7 inhibited the migration and invasion of ovarian cancer cell and reversed epithelial-mesenchymal transformationTranswell assay showed that the cell migration and invasion abilities of the sh-BMP7 group were significantly suppressed compared with that of the sh-NC group.Analysis using western blotting indicated that the decreased expression of BMP7 in A2780 cell exhibited significantly increased expression of E-cadherin,while the expression levels of N-cadherin and Vimentin were reduced.Moreover,the expression levels of Snail and Slug were also decreased.6.Down-regulation of BMP7 enhanced the sensitivity of ovarian cancer cell to paclitaxelAccording to the CCK-8 experiment result,when the cell was treated with paclitaxel at different concentrations for 24h,the relative survival rate of the sh-BMP7 group was lower than that of the sh-NC group at the same concentration of paclitaxel(P<0.05).Furthermore,the result of flow cytometry indicated that the rate of early apoptosis of cell in the sh-BMP7 group was significantly higher than that of the sh-NC group after 24h of paclitaxel treatment at a concentration of 2?M(P<0.05).7.Down-regulated BMP7 resulted in decreased expression levels of Smad 1/5/9 p-Smad 1/5/9 and 1D2Western blotting result showed that the expression levels of Smad 1/5/9,p-Smad 1/5/9 and ID2 in sh-BMP7 group were decreased compared with those in sh-NC group.The ID2 mRNA level of sh-BMP7 group was decreased compared with sh-NC group by RT-qPCR(P<0.05).Immunofluorescence result showed that the expression of p-Smad 1/5/9 protein in sh-BMP7 group was decreased.ConclusionsBMP7 is overexpressed in ovarian cancer tissues.Knocking down BMP7 in ovarian cancer A2780 cell inhibited cell proliferation,migration and invasion,reversed epithelial-mesenchymal transformation,led to cell cycle arrest and increased cell sensitivity to paclitaxel,and BMP7 plays these roles in ovarian cancer cell perhaps through the BMP7/Smad 1/5/9 pathway.BMP7 may be a potential therapeutic target for improving the efficiency of ovarian cancer treatment.