Expression Of Basigin Alternative Splicing Variants And Their Functions In Epithelial Ovarian Cancer

BackgroudOvarian cancer is the most lethal gynecologic malignancy among women in manycountries. Epithelial ovarian cancer is the most common ovarian cancer and accounts forapproximately70%of all ovarian malignant diseases. It is the sixth most common cancerand the fifth leading cause of cancer-related death in women worldwide. Being largelyasymptomatic, almost70%of cases are at an advanced stage at diagnosis. Despitesignificant advances in surgery and chemotherapy over the last few decades, therapeuticfailure and disease progression are still quite frequent. The five-surivival rate is still low.Therefore, there is an urgent requirement for new biomarkers for ovarian cancer, so as todevelop better early diagnostic andtherapeutic strategies.Basigin, also known as CD147, extracellular matrix metalloproteinase inducer (EMMPRIN), neurothelin,5A11, gp42, OX-47, and CE9, is a widely expressed integralplasma membrane glycoprotein that belongs to the immunoglobulin superfamily. It is ahighly glycosylated58-kDa transmembrane protein that has four isoforms. Basigin hasbeen shown to induce the production of several Matrix metalloproteinases (MMPs) whichplay an important role in tumor invasion and metastasis formation. It turned out to bemultifunctional and involved in various physiological processes such as fetal development,reproduction, T cell differentiation, and neural and retinal functions. Increased expressionof Basigin has been found in several human tumors suggesting a role in humancarcinogenesis. Moreover, it has been shown that higher expression of Basigin wasassociated with poor prognosis of cancer patients and could serve as an independentpredictor of poor survival in cancer. The most life-threatening aspects of the oncogenicprocess are invasion and metastasis.There is a few study about Basigin which has four splice variants, and how itsexpression and function in ovarian cancer is one of the forefront of research in relatedfields. This is the first report that has examined the expression patterns of Basigin and itsisoforms in ovarian cancer tissues and cell lines. We also investigated the correlationsbetween Basigin-2expression and various clinic pathologic parameters, and its prognosticvalue for survival of patients with epithelial ovarian cancer. Furthermore, we determinedthe functional roles of Basigin-2in ovarian cancer cell lines. The results presented herehelp to evaluate the suitability of Basigin-2as therapeutic target.Objective1. To study Basigin-2spliced variant expression in the epithelial ovarian cancer.2. To study the relationship between Basigin-2protein expression and clinical pathology,prognostic value in epithelial ovarian cancer.3. To study the potential correlation between Basigin and invasion and metastasis inovarian cancer cells.Methods1. RT-PCR, Real-time PCR and Western-blot were used to explore Basigin-2, Basigin-3and Basigin-4expression in ovarian cancer cell lines and tissues.2. The relationship between Basigin-2expression and clinical pathology, prognosticvalue of ovarian cancer.a) Immunohistochemistry staining was performed to investigate Basigin-2expression ina total of146ovarian tissue specimens.b) Kaplan Meier analysis and Cox proportional hazards model were applied to assess therelationship between Basigin-2and progression-free survival (PFS) and overallsurvival (OS).3. To evaluate possible contributions of Basigin-2to MMP secretion and cellmigration and invasion, the overexpression vectors pcDNA3.1-Basigin-2andBasigin-2-siRNA were transfected into HO-8910and HO-8910PM cellsrespectively.Results1. The expression of Basigin splicing variants in the ovarian cancer ovarian tissuespecimens and ovarian cancer cells.a) RT-PCR was performed to determine whether Basigin mRNA variants were expressedinovarian cancer cell lines. The results indicated that Basigin-2, Basigin-3andBasigin-4were detectable in all these five cell lines.b)27normal and71tumor samples were used to analyze Basigin-2, Basigin-3andBasigin-4mRNA expression by real-time quantitative PCR assay. The resultsindicated that mRNA levels of Basigin-2and Basigin-4were significantly differentbetween the cancer and normal samples. Expressions of Basigin-2and Basigin-4mRNAs in ovarian normal tissues were lower than that in cancer tissues (P<0.001and P=0.0129, respectively), whereas mRNA level of Basigin-3showed nosignificant difference between ovarian normal and cancer tissues (P=0.2703).c) We used real-time PCR analysis to characterize mRNA expression of Basigin-2,Basigin-3and Basigin-4in five ovarian cancer cell lines. Expression of Basigin-2andBasigin-4mRNA in HO-8910cell line was lower than that in its subline cell line HO-8910PM,(P<0.05). But mRNA level of basigin-2was much higher thanbasigin-4in ovarian cancer (>100fold to basigin-4). Expression of Basigin-3mRNAshowed no significant difference between HO-8910cells and HO-8910PMcells (P>0.05).d) We used western blot analysis to characterize protein expression ofBasigin-2in fiveovarian cancer cell lines. Basigin-2was detectable in all these five cell lines, withhighest expression in HO-8910PM cells. Expression of Basigin-2protein inHO-8910cell line was lower than that in its subline cell line HO-8910PM (a highlymetastatic cell line derived from HO-8910).2. The relationship between Basigin-2expression and clinical pathology, prognosticvalue of ovarian cancer.a) We described the relationship between basigin-2expression and clinicopathologicalparameters of146cases of epithelial ovarian cancer with immunohistochemicalanalysis, and was localised in the cytomembrane of tumor cells.b) Basigin-2expression was positively correlated with lymph-vascular spaceinvolvement (P=0.012), lymph node metastasis (P=0.017), but not with age, FIGOstage, degree of differentiation, or Histological type (P>0.05).c) High basigin-2expression was associated with a significantly shorter overall survivaland progression-free survivalin epithelial ovarian cancer.d) By using univariate Cox proportional analysis, Basigin-2expression was statisticallycorrelated to progression-free survival (P<0.001) and overall survival (P=0.002).e) By using multivariate Cox proportional analysis, considering statistically significantvariables and Basigin-2expression and FIGO staging were independent factors ofprogression-free and overall survival prognosis predictions.3. To evaluate possible contributions of Basigin-2to MMP secretion and cellmigration and invasion, the overexpression vectors pcDNA3.1-Basigin-2weretransfected into HO-8910cells.a) After transfection of cells, western blot analysis was performed to analyze theexpression levels of Basigin-2protein. The expression of Basigin-2protein was increased in pcDNA3.1-Basigin-2transfected cells compared with pcDNA3.1transfected cells and untransfected cells (P<0.05).b) We employed gelatin zymography to analyze the effect of Basigin-2overe xpressionon MMP-2and MMP-9enzyme activities. We observed that MMP-2and MMP-9gelatinase activities in the culture media of pcDNA3.1-Basigin-2-transfectedHO-8910cells were substantially higher than in cells transfected with control cells,(P<0.05).c) HO-8910cells that were transfected with the pcDNA3.1-Basigin-2plasmid exhibiteda significant increase in cellular migration as compared with control cells (P<0.05).d) HO-8910cells transfected with pcDNA3.1-Basigin-2displayed much higher invasivecapability compared with control cells as evidenced by increased number of cellsinvading through the matrigel (P<0.05).4. To evaluate possible contributions of Basigin-2to MMP secretion and cellmigration and invasion, the Basigin-2siRNA were transfected into HO-8910PMcells.a) After transfection of cells, western blot analysis was performed to analyze theexpression levels of Basigin-2protein. The expression of Basigin-2protein wasdecreased in Basigin-2siRNA-transfected cells compared with controlsiRNA-transfected cells and untransfected cells (P<0.05).b) We observed that MMP-2and MMP-9gelatinase activities in the culture media ofBasigin-2siRNA-transfected HO-8910PM cells were substantially lower than in cellstransfected with control cells (P<0.05).c) The results showed a significant reduction in motility of Basigin-2siRNA-transfectedHO-8910PM cells compared with the control groups (P<0.05).d) We determined the ability of Basigin-2siRNA-transfected cells to invade throughMatrigel in Transwell chambers. A significant reduction in the number of invadingcells was observed for siRNA-transfected cells after24h compared with the controlcells (P<0.05). Conclusion1. This study is the first to examinethe expression patterns of Basigin and itsisoforms(Basigin-2、Basigin-3、Basigin-4) in ovarian cancer tissues and cell lines. Theresults indicated that mRNA levels of Basigin-2and Basigin-4were significantlydifferent between the cancer and normal samples, and mRNA level of Basigin-2wasmuch higher than Basigin-4in ovarian cancer. This suggests that Basigin-2plays animportant role in ovarian cancer.2. Our results indicated that high Basigin-2expression was significantly correlatedwithlymph-vascular space involvement and lymph node metastasis, suggesting that itsexpression might be important for the acquirement of malignant potential in epithelialovarian cancer.3. Basigin-2expression was significantly associated with both overall survival andprogression-free survival. These results led us to believe that Basigin-2might be usedas a candidate biomarker for epithelial ovarian cancer diagnosis and therapy.4. The result of experiment showed Basigin-2can promote ovarian cancer cell migration,invasion, and play a more important function in the progression of ovarian cancer.