The Effect Of SPARC Gene Over-Expression On Biological Characteristics Of Human Ovarian Carcinoma Cells With Directional High Lymphatic Metastasis

Chapter I Construction and identification of lentiviral vector carrying SPARC geneObjective:For further study the function and mechanism of secreted protein acidic and rich in cysteine gene (SPARC), a lentiviral vector expression the gene of SPARC was constructed and lentivirus particles were infected into human ovarian carcinoma cells with directional high lymphatic metastasis cells (SKOV3-PM4) and then the corresponding expression products were identified. Methods:Full-length of the SPARC gene was amplified by PCR and cloned into plasmid pLV-EGFP. Then it was further confirmed by PCR and sequencing; the recombinant pLV-EGFP and packaging plasmid co-transfected the virus packaging 293 T cells in proportion, SKOV3-PM4 cells were infected with the virus particles; the transfected cells were selected by puromycin; then the SPARC gene was detected by Real-time PCR and its protein expression was detected by Western blot, and the protein distribution was observed by cell immunofluorescence under laser confocal microscopy. Results:The recombinant expression vector was successfully constructed, and lentiviruses were successfully packaged by the 293T cells. The cells that expressed target gene was efficient and stable. And SPARC gene was up-regulated significantly at both mRNA and protein level (P<0.05). Cell immunofluorescence results showed SPARC is in the perinuclear and the cytoplasm. Conclusion:A lentiviral expression system of SPARC was successfully constructed and lentivirus particles were infected into target cells to upregulate the expression of SPARC gene, which is an experimental basis for exploring its biological function in lymph node metastasis of ovarian cancer.Chapter II The Effect of SPARC Gene Over-expression on proliferative ability and metastasis of Human Ovarian Carcinoma Cells with Directional High Lymphatic MetastasisObjective:The aim of this study was to investigate the effect of SPARC over-expression on proliferative ability and metastasis of SKOV3-PM4 cells. Methods:The proliferative ability of SKOV3-PM4 cells was detected by cell counting method and colony formation test, and the cell cycle was measured by flow cytometry. The abilities of cell invasion and migration were examined by wound healing assay and Transwell assay. Results:Cell growth curve and cell colony formation showed that the proliferative ability of SKOV3-PM4-SPARC cells was significantly inhibited (P<0.05). There were no significant differences in cell cycle distribution. The invasion and migration capabilities of SKOV3-PM4-SPARC were also significantly decreased (P<0.05). Conclusion: The abilities of proliferation and invasion of epithelial ovarian cancer cells with directional high lymphatic metastasis were inhibited by SPARC gene overexpression. SPARC gene may play an important role in biological function as a tumor suppressor gene.Chapter III The Effect of SPARC Gene Over-expression on VEGF family signaling pathway of human ovarian carcinoma cells with directional high lymphatic metastasisObjective:The study aimed at investigating the effect of SPARC gene over-expression on VEGF family signaling pathway of SKOV3-PM4 cells. These changes provided the theoretical basis for the therapeutic methods of lymph node metastasis in ovarian cancer. Methods:The expression of VEGF family signaling pathway was detected at protein level by Western blot to observe the effect of SPARC over-expression. The protein expression level of SPARC in transplanted tumors in nude mice and ovarian malignant tumors were detected by immunohistochemistry staining, and IHC staining was used to explore the correlation between SPARC and VEGF-C, VEGF-D, LVD, MVD. Results:The results of Western blot revealed that compared with the untransfected cells, the expression of VEGF-D and PIGF were significantly reduced at protein level (P<0.05); Immunohistochemistry staining showed the positive expression rate of SPARC in ovarian malignant tumors with lymphatic metastasis and ovarian malignant tumors with no lymphatic metastasis were 15.00% and 48.15% respectively (P<0.05), but there was no significant difference between the transplanted tumors of SKOV3 and SKOV3-PM4 (P>0.05). The negative correlations of expression level were found between SPARC and VEGF-C, VEGF-D protein (P<0.05), and the inverse correlations of SPARC and LVD, MVD were found in ovarian malignant tumors (P<0.05). Conclusion:SPARC gene is closely associated with ovarian cancer lymph node metastasis, which may play an important role in biological function as a tumor suppressor gene, and SPARC gene has a certain impact on angiogenesis and lymphangiogenesis. Therefore, the further study on SPARC gene may help to illustrate the mechanism of lymph node metastasis in ovarian cancer.ChapterlV The latest research on the relationship between SPARC gene and the tumorigenesis and tumor progressionSecreted protein acidic and rich in cysteine (SPARC) is a kind of small molecule secretory matrix protein and plays a role in biological function of cell proliferation, invasion, metastasis and angiogenesis. This article just reviewed present status of relation between the biological characteristics of SPARC gene and tumors and focused on the role in tumorigenesis and progression.