Detection Of Rab25 In Ovarian Cancer And Research On Induced Apoptosis Of Ovarian Cancer Cell By Gene Silencing

Ovarian cancer is a leading cause of death among gynecologic malignancies. In spite of advances in surgery and chemotherapy, its mortality rate remains relatively unchanged during recent several decade. The current optimum approach to therapy consists of cytoreductive surgery followed by combination chemotherapy. Clinical trial have established that carboplatin plus a taxane(usually paclitaxel) can be considered to be the treatment of choice for meet patients with advanced disease. Most patients will achieve a clinical complete remission with such a combination. However the median time to progression is less than 2 years, and for patients with optimal stage III disease, median survival will be approxi- mately 5 years. Therefore, new adjunctive therapy is of great importance.Rab proteins are Ras-like small GTPases, which have crucial roles in vesicle trafficking, signal transduction, and receptor recycling which in turn regulate normal cellular activity. Recent studies have shown multiple links between Rab GTPase dysfunction and associated regulatory proteins in human diseases including cancer, and these changes are closely correlation with development of numerous diseases. The study have recently shown that Rab25, located at chromosome 1q22, is amplified at the DNA level and overexpressed at the RNA level in ovarian and breast cancer. These changes correlated with a worsened outcome in both diseases. In addition, enforced expression of Rab25 in both breast and ovarian cancer cells decreased apoptosis and increased proliferation and aggressiveness in vivo, potentially explaining the worsened prognosis. So, a better understanding of genetic alterations as well as the physiologic and pathophysiologic roles of Rab GTPases may open new opportunities for therapeutic intervention and better outcomes.With molecular biology technology gradually mature, gene therapy as a way that specificity block or regulate virulence gene is becoming new therapeutic way of ovarian cancer. RNA interference(RNAi) technology may be a optimized means with following important features: High stability, High efficiency, High specifi- city. Hence, it may become a new means of tumor gene therapy.ObjectiveTo investigate the expression of Rab25 in ovarian cancer tissue and the inhibition of Rab25 gene expression by RNA interference technology, discuss the mechanism of action of Rab25 on ovarian cancer.MethodsPart One The expression and significant of Rab25 in ovarian malignant tumor Immunohistochemical method was employed to detect the expression of Rab25 in normal ovary, carcinoid and carcinoma ovarian tissues, then analyzed with regard to clinical stage, pathological, histology type and metastatic.Part Two The study of inhibiting the expression of Rab25 gene in ovarian cancer by RNA interference and pathway of action.Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cells. The Rab25 siRNA eukaryotic expression vectors pSUPER /Rab25 siRNA was constructed by Using RNAi technology. The direction was confirmed by endonuclease digestion. Using lipidosome method, the Rab25 highly expressed human malignant ovarian cells (A2780) were transfected with the siRNA recombinant vector. Stable clones cells were obtained after G418 screening. To observe the biologic behavior of ovarian cancer cells.一,Construction of Rab25 siRNA eukaryotic expression vectors and the confirmation on the transfected cellsAccording to Rab25 mRNA sequence in the Genebank,a pair of 64-nt oligonucleotides, each containing the sites of restriction endonuclease at both ends, were designed and synthesized. Oligonucleotides were annealed and ligated with linearized pSUPER by T4DNA ligase. The recombinants (named pSUPER /Rab25 siRNA ) were finally sequenced and identified by enzyme cutting and sequencing. RT-PCR assay,indirect immunofluore- scence and Western blot studies testified that the protein and mRNA level.二,The role of Rab25 Expression in ovarian cancer cellsKnockdown the expression of Rab25 in ovarian cancer cells, we observed the change of cells proliferation and apoptosis, invasion and adhesion, morphology and so on.1. Morphological observations of Rab25 siRNA in ovarian cancer cells Inverted microscope and electron microscope were used to observe changes in cell morphology, especially dendron, cell body and typical apoptotic cell.2. Effect on the proliferation and apoptosis of Rab25 siRNA in ovarian cancer cellsMTT was performed to test effectdof different ovarian cancer cells, OD readings were obtained using an autoreader at 490 nm, draw Growth curve; Flow cytometry analysis was used to determine apoptosis and cell cycle of the cells; Apoptosis index of ovarian cancer cells was tested by TUNEL.3. Effect on the invasion and adhesion of Rab25 siRNA in ovarian cancer cellsTo detect the abiliry of adhesion among ovarian cancer cells by cell-adhering method; to detect the ability of invading in vitro by transwell-ECM method and wound-healing method.4. The erecting of nude mice model of transplanted ovarian cancer celland the effect on the model of Rab25 siRNAWe erected the model on subcutaneously implanted tumor in nude mice. 106 ~ 2×106 cells were injected subcutaneously into 4- to 6-week-old athymic female nude mice. The mice were kept in pathogen-free environments and checked every 2 days for about 1-1.5 months. The date at which grossly visible tumor first appeared and the size of the tumor were recorded. The mice were killed when tumors reached 2.5 cm in diameter.三,The initial study of mechanism of action on Rab25 To detect the expression of Bcl-2 and Bax by immunohistochemistery,RT-PCR,Western blot, and to investigate the pathway of Rab25.Reults1. Rab25 protein evels were high in ovarian borderline epithelial tumors and carcinoma, and it is low in normal ovary and benign tumors, the difference was significant(P<0.05). The expression of Rab25 was correlated with lymph node metastases and TNM stages. It suggested that the high level of Rab25 mignt play an important role in the carcinogenesis, development and prognosis of malignant tumors.2. Rab25 siRNA expression vector was successfully constructed and identified by double endonuclease digestion. Sequence analysis of inserted fragment revealed the same sequence as synthesized siRNA oligonucleotides, and achieved continuous siRNA expression in A2780 cells by gene transfection. The result of RT-PCR, indirect immunofluorescence and Western blot showed that, the efficiency of inhibition was related to target selecting. Under light microscope, they displayed large epithelioid perikaryon and stubby dendrites with occasional multidendricity as opposed to slender perikaryon and bipolar dendrites of control cells, and under electron microscope we saw typical apoptosis cells. The alteration in cell morphology suggested abnormal metabolization in Rab25 siRNA transfected cells. Clone-forming ability decreased, the proliferation of Rab25 siRNA transfected cells were inhibited compared with the control cells by using MTT assay. Compared with the controls, Rab25 siRNA transfected cells were retarded in G1 stage and the percent of cells in G1 stage increased significantly. In order to further confirm the apoptosis, flow cytometer detection was used. After the double stain of PI and annexin V FITC, more apoptosis cells could be seen in the siRNA transfected cell group. The difference of annexin V FITC positive cells between the Rab25 siRNA transfected cell groups and the control cells were significant. Statistics showed a significant growth suppression of Rab25 siRNA vector transfected cells. TUNEL showed apoptosis cells obviously. The A2780 cells that transfected Rab25 siRNA vector displayed descended invasiveness, adhesion and metastasis ability in vitro in comparison with the control cells by cell-adhering and transwell-ECM method. Nude mice model with implanted tumor were built successfully, after observeing 30-45 days, the tumors of Rab25 siRNA vector transfected cells groups was much more smaller than those of the control groups. This suggested that Rab25 siRNA suppressed tumor formation in vivo in nude mice xenografts. The results showed that compared with control groups, from mRNA level to protein level, the expression of Bcl-2 significant decreased; the expression of Bax significant increased by RT-PCR,immunohistochemistry and Western blot detection, These show that Rab25 induce cell apoptosis by regulating the level of Bcl-2/Bax.Conclusion1. In malignant tumors, the expression of Rab25 protein was high by using immunohistochemical methods, and the expression of Rab25 was correlated with TNM stages.2. The high expression of Rab25 protein participated in metastasis of ovarian cancer. 3. Rab25 siRNA can interfere with the protein and mRNA expression of Rab25 in ovarian cancer cells and inhibit their growth and proliferation, enhance apoptosis, inhibit the ability of invasion and adhesion, inhibit tumor growth.4.Rab25 siRNA can inhibit growth and invasion of ovarian cancer by induce cell apoptosis signal passway.