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BackgroundOvarian cancer, a serious threat to the health of women, is the leading cause of death from gynecologic malignancies with a dismal poor overall 5-year survival rate (30%). With appeared insidiously, lacking of effective screening methods in early stage , especaily proned to invasion and metastasis, so most patient were diagnosed in late stage, seriously affecting the prognosis of patients with ovarian cancer. Among them, invasion and metastatasis were to become the bottleneck for curing it. Currently, gene therapy has become the hotspot in the treatment of ovarian cancer research , and in animal experiments and theⅠ,ⅡandⅢclinical study it achieved a certain effect, which is expected to become a new treatment model following the surgery, radiotherapy and chemotherapy. It was reported that , as one of the tumor metastasis suppressor gene, BRMS1 gene could inhibit a variety of tumors invasion and metastasis, including ovarian cancer. But because of its role is complex and atypical, and still less research on the specific mechanism , so in the cellular level studying the effect and possible mechanism of BRMS1 gene on the invasion and metastasis of ovarian cancer cells in vitro , could explore a new target to control ovarian cancer invasion and metastasis for gene therapy, still more, clinical theoretical foundation will be established, so it will have certain scientific significance and potential clinical applications.ObjectiveUsing RNA interference technology, whit transient transfection, BRMS1 gene was silenced, by observing the changes of human ovarian cancer OVCAR3 cell invasion and metastasis , and detecting the expression changes of tumor metastasis-related protein NF-kB p65, OPN and uPA, to investigate the effect and possible mechanism of BRMS1 on the invasion and metastasis of human ovarian cancer cells.Methods1) Culture the human ovarian cancer OVCAR3 cellsOVCAR3 cells were cultured with Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO) supplemented with 10% fetal bovine serum(FBS), 1% penicillin and streptomycin, in 5% CO2 and 95% air at 37℃. Transfection experiments began when adherent cells growed to 80% -90% confluent.2) Using of RNA interference, build BRMS1 gene silenced human ovarian cancer OVCAR3 cell lineBased on the gene sequence of GenBank, three BRMS1-siRNA target sequence, and a negative control sequences ( NC-siRNA) were designed, synthesized by biotechnology company. Ovarian cancer cells were randomly divided into 3 groups: experimental group with BRMS1-siRNA, negative control group with control siRNA and blank control group. Mixture of siRNA-liposome duplexes were transiently transfected into OVCAR3 cells using Lipofectamine 2000 (Invitrogen) according to the instructions from manufacturer. When 48 hours after transfection, total cellular RNA and total protein extracted, by real-time fluorescence quantitative PCR and western blot respectively, analysing mRNA and protein levels to screen and identify the most efficient siRNA for the follow-up experiment.3) Changes of human ovarian cancer cells invasion and metastasis detecting by Transwell invasion and migration assayIn vitro cellular invasion and migration (no matrigel) assays were performed by determining the ability of cells to invade through a synthetic basement membrane (Matrigel, BD Biosciences). Briefly, transfected OVCAR3 cells (5×104 per well) were plated to the top chamber in serum-free media and incubated with serum-containing media (10%) as a chemoattractant in the bottom chamber. Cells were then incubated for 24 h. After incubation, filters were fixed and stained with Crystal Violet Staining Solution. Noninvading cells were removed and invaded cells on the underside of the filter were enumerated using an inverted microscope.4) Changes of NF-kB p65, OPN and uPA expression detecting by Western blot , immunocytochemistry or immunofluorescence assayThe differences were detected in the expression of NF-kB p65, OPN and uPA by Western blot, immunocytochemistry or immunofluorescence, quantitative and qualitative analysises were detected the expression of these metastasis-related proteins when before and after BRMS1 gene silenced.5) Statistic analysisResults were calculated as mean±SD((χ|-)±s). SPSS 16.0 was used for all analysis, and P <0.05 was considered statistically significant.Results1. Select the highest efficiency BRMS1-siRNA, and gene silencing efficiency as high as 82.3%, which was laid the basis to construct retroviral vectors for stable transfection.2. BRMS1-siRNA transfected to human ovarian cancer OVCAR3 cells, cell invasion and metastasis significantly increased compared with two control groups (P <0.05).3. BRMS1-siRNA transfected to human ovarian cancer OVCAR3 cells, tumor metastasis associated protein NF-kB p65, OPN, and the expression of uPA were detected by Western blot, immunocytochemistry or immunofluorescence with qualitative and quantitative analysis, we found that the experimental group were significantly higher than the two control groups (P <0.05).ConclusionBRMS1 inhibits expression of uPA and OPN through reduction of NF-κB subunit, p65 protein. In turn, this leads to inhibit ovarian cancer cell invasion and metastasis. This study unveils a potential novel mechanism by which BRMS1 inhibits metastasis of ovarian cancer.
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