The Regulation Of ShRNA Targeting BRMS1on The Invasion And Metastasis Of Human Ovarian Cancer Cells

BackgroundOvarian cancer is one of the most common malignant tumors in women, which isranking the first among the leading gynecologic cancer. About70%of patients have localor distant metastases before they seek medical attention due to lack of typical symptomand effective tests to make a diagnosis, as a result, five-year survival rate of patients withan advanced disease is still dissatisfied. Relapse and metastasis always result in poorclinical outcome. A better understanding of the metastatic mechanisms of ovarian cancer istherefore needed to determine effective therapeutic interventions to either eradicate orslow metastatic outgrowth.Many factors are involved in regulating metastasis through diverse mechanisms.Among metastasis suppressors, breast cancer metastasis suppressor1(BRMS1) couldinhibit a variety of tumors invasion and metastasis, including ovarian cancer. Recentevidence indicate that BRMS1inhibits metastasis in breast cancer cells via reducingCXCR4and IL-6expression and promoting ING4expression; however, the functionalimplications of BRMS1and its relationship to the target genes in ovarian neoplasms arenot clear. Therefore, we investigated the potential mechanisms of BRMS1-mediatedmetastasis suppression in ovarian cancer, and found a potential new therapeutic target. ObjectiveTo construct a plasmid containing a short hairpin RNA(shRNA) against BRMS1andtransfect it into the ovarian cancer cell line OVCAR3, and then to observe the influence ofBRMS1knockdown on cell adhesion, migration, invasion and angiogenesis.Methods1. Screening of ovarian cancer cell line with high expression of BRMS1Real time PCR and Western blot were used to detect the BRMS1mRNA and proteinin ovarian cancer cell lines ES-2, HO-8910, SKOV3and OVCAR3.2. Plasmids construction and identificationThe BRMS1-siRNA template DNA sequence for shRNA was designed andsynthesized. The annealed siRNA template was inserted into pGPU6/GFP/Neo plasmid.The recombinant plasmid of BRMS1-shRNA was identified by restrictive enzymedigestion and sequence analysis.3. Cell transfection and clones screenAccording to the manufacturer，s protocol for Lipofectamine2000,pGPU6/GFP/Neo-BRMS1or pGPU6/GFP/Neo-NC were transfected into OVCAR3cells,and the stably transfected cells were obtained after being screened with G418. Then thecells were visualized under fluorescence microscopy. Real-time PCR and Western blotwere applied to analyze BRMS1mRNA and protein levels, respectively.4. Effect of BRMS1knockdown on the metastasis ability of ovarian cancer cellsCell adhesion was measured by cell adhesion assay. Cell invasion and migration weremeasured by Transwell invasion and migration assay. Angiogenesis capacity of HUVECsco-cultured with OVCAR3cells was determined by tube formation assay.5. Mechanism of BRMS1on the invasion and metastasis of ovarian cancer cellsReal-time PCR was utilized to detect the expression of CXCR4mRNA. Westernblot was used to detect the expression of ING4and IL-6. Furthermore, an electrophoreticmobility shift assay (EMSA) was applied to explore whether BRMS1regulates CXCR4expression through the NF-κB pathway.6. Statistical analysisAll experiments were performed at least in triplicate and data were compiled from three separate experiments. The results were calculated as means±SD. All statisticalanalyses were determined by one-way ANOVA using the SPSS16.0software. AP-value<0.05was considered significant.Results1. A cell line with high expression of BRMS1, OVCAR3, was screened successfully.2. The accurancy of the constructs was confirmed by restriction endonuclease digestingand DNA sequencing.3. After the stable transfection of BRMS1-shRNA, the expression levels of BRMS1mRNA and protein were measured by real-time PCR and Western blot analysis,respectively. The BRMS1mRNA level declined significantly in the BRMS1-shRNAtransfected cells, with an average inhibition of85.15%compared to the blank controlgroup（P<0.01）. BRMS1protein expression was also decreased, with an averageinhibition of46.67%in the BRMS1-shRNA group（P<0.01）.4. Inhibition of BRMS1markedly enhanced cell adhesion, invasion, migration and theangiogenic capacity.5. Real-time PCR revealed that CXCR4mRNA was1.78-fold higher in BRMS1-shRNAtransfected cells. Western blot analysis revealed that the protein levels of CXCR4andIL-6were elevated by1.26-fold and1.5-fold, respectively, while BRMS1silencing ledto30%decreased of ING4protein expression（P<0.01）.6. The EMSA results suggested that BRMS1knockdown increase the DNA bindingactivity of NF-κB to the CXCR4promoter, compared to the control groups, whereasthe unlabeled competitive sequence markedly inhibited this binding.Conclusion1. pGPU6/GFP/Neo-BRMS1plasmid was successfully constructed and could effectivelysuppress BRMS1expression at both the mRNA and protein levels in OVCAR3cells.2. After the down-regulation of BRMS1, the adhesion, invasion, migration andangiogenesis of OVCAR3cells were significantly promoted.3. Knockdown of BRMS1obviously up-regulated the expression of CXCR4and IL-6,while decreased ING4expression. Our data elucidated that these metastasis-relatedgenes may involve in the process of BRMS1regulating metastatic potential in ovarian cancer.4. Knockdown of BRMS1in ovarian cancer cells was associated with upregulation ofCXCR4mediated by NF-κB activation, which then increased the metastatic potential.