A Study Of The Role Of BRMS1 In Breast Cancer Cells And Its Mechanisms

Objective:to investigate the function of BRMS1 and to explore its mechanisms of action in human breast cancer cells.Methods:The entire open reading frame(ORF) of human BRMS1 gene was amplified by RT-PCR and cloned into pcDNA3.1(+) vector.The recombinant vector was identified by restriction enzyme digestion and automated sequencing analysis then transfected into MDA-MB-231 with high capability of spontaneous pulmonary metastasis cells(2m4) by stable transfection. The pcDNA3.1(+)-BRMS1 positive and the empty vector positive colonies were identified by RT-PCR.In vitro,cell morphology,cell growth rate, cell cycle distribution,apoptosis,migration and invasion were detected respectively by light microscope and transmission electron microscope, CCK-8 assay,FCM with PI staining,FCM with Annexin V/PI staining,wound healing and three-dimensional cell migration assay and in vitro invasion assay.To further examine the effects of BRMS1 overexpression on tumor growth and metastasis,we performed the in vivo assay using an orthotopic xenograft tumor model in the athymic mice.We examined the primary tumor growth in mammary fat pad and the pulmonary metastasis by H&E section. To explore the BRMS1mechanisms of action,the effect of pertussis toxin on BRMS1-induced inhibition of cell migration was tested by three-dimensional cell migration assay.Determination of intracellular cAMP was preformed by a radioimmunology method.The changes of the gap-junctional Cx26,Cx32,Cx43 mRNA expression were examined by RT-PCR. To investigate the effect of BRMS1 overexpression on chemotherapeutic drug sensitivity of tumor cells.The drugs 5-fluorouracil,adriamycin, docetaxel were selected.The proliferation inhibition rate of breast cancer cells was analyzed by CCK-8 assay. Results:The pcDNA3.1(+)-BRMS1 vector was recombined successfully.In vitro,BRMS1 overexpression changed cell morphology.Cells had a tendency to become wrinkled and round.The numbers of pseudopodium and microvilli were reduced.However BRMS1 overexpression had little effect on cell growth rate,cell cycle distribution and apoptosis,it could significantly reduced cell migration and invasion.In vivo,BRMS1 transfectants had similar volume of primary tumor in comparasion to mock transfected and parental cells at the end of the assays The rate of pulmonary metastasis in nude mice was dramatically decreased by BRMS1 overexpression.Furthermore,the number and volume of metastasis was significantly reduced.In terms of BRMS1 mechanisms of action,BRMS1's effect on cell migration was abolished after PTX pretreatment.Meanwhile, BRMS1 overexpression increases intracellular cAMP.On the other hand, connexins expression profiles were changed through increasing expression of Cx26 by BRMS1 overexpreesion.In terms of chemotherapeutic drug sensitivity,BRMS1 overexpression had little effect on chemotherapeutic drug sensitivity of tumor cells for selected drugs.Conclusion:BRMS1 reduces the metastatic propensity of breast cancer cells in vivo without affecting their tumorigenesis.It has no effect on cell growth rate,cell cycle distribution,apoptosis,but it can change cell morphology,reduced cell migration and invasion.The G-protein-coupled cAMP signaling pathway may have a important role in the metastasis of breast cancer cells that can suppressed by BRMS1.BRMS1 also changes connexins expression profiles through increasing expression of Cx26 in breast cancer cells to inhibit metastasis.