Effects of Breast Cancer Metastasis Suppressor 1 Gene on Angiogenesis of Human Ovarian Cancer Cell and Its Mechanism

Objective To investigate the effects and mechanism of breast cancer metastasis suppressor 1(BRMS1) on angiogenesis in human ovarian cancer cell line OVCAR3. Methods Vector containing a short hairpin RNA(shRNA) against BRMS1 was constructed and the positive clone was named pGPU6/GFP/Neo-BRMS1.The BRMS1 shRNA or a non-specific sequence(as the negative control) was transfected into OVCAR3 cells by LipofectamineTM2000 and stably transfected cells were obtained by screening with G418.The expressions of BRMS1 mRNA and protein in OVCAR3 cells were performed using real time PCR and Western blot,respectively.Angiogenesis capacity of human umbilical venous endothelial cells(HUVECs) co-cultured with OVCAR3 cells was determined by tube formation assay.Furthermore,the expressions of inhibitor of growth 4(ING4) and Interleukin-6(IL-6) were detected by Western blot. Results The recombinant plasmid could be successfully transferred into OVCAR3 cells.Real time PCR and Western blot analyses demonstrated that BRMS1 expression was efficiently down-regulated.Stable suppression of BRMS1 in human ovarian cancer cells significantly promoted the ability of HUVECs to form tubular structures in the co-culture,and the number of tubules was markedly increased in transfection of BRMS1 shRNA than that in the blank control group or negative control group.Moreover,the protein level of ING4 in interference group was decreased by 30%,while BRMS1 knockdown led to 1.5 fold increase of IL-6 protein expression(P<0.05). Conclusion Knockdown of BRMS1 may play a critical role in promoting the angiogenesis-inducing ability of ovarian cancer cells,and BRMS1 regulates metastatic potential at least in part through the regulation of ING4 and IL-6.