| Objective:In this study, we aimed to detect the expression of Nedd4 L in ovarian epithelial carcinoma and its regulation mechanism on cell proliferation, migration, invasion, apoptosis and cell cycle in SKOV3 cell, to explore a new target in treating the ovarian cancer. Method:1. To detect the expression of Nedd4 L in normal ovarian tissue, benign ovarian tumor tissue and ovarian cancer tissue by immunohistochemical technique; 2. To analyze the relationship between Nedd4 L expression and clinicopathological features of ovarian cancer patients by chi-square test; 3. The expression of Nedd4 L was analyzed by Kaplan-Meier method. 4. The expression of Nedd4 L in the human ovarian cancer cell line SKVO3 was induced by Lipofectamine 2000 and pc DNA3.1(+)-Nedd4 L. And the expression of Nedd4 L in ovarian cancer cells was detected by real-time quantitative PCR, immunohistochemical staining and immunocytochemistry. 5. The eukaryotic expression vector pc DNA3.1(+)-Nedd4 L containing Nedd4 L was transfected into human ovarian cancer cell line SKVO3, scratches and MTT were used to determine the cell viability. 6. The eukaryotic expression vector pc DNA3.1(+)-Nedd4 L containing Nedd4 L was introduced into human ovarian cancer cell line SKVO3, To observe the effect of Nedd4 L on cell cycle and apoptosis of Nedd4 L cells after transfection with Nedd4 L recombinant vector; 7. To observe the growth of Nedd4 L transfected nude mice in vivo. Result:1. NEDD4 L were expressed in the cytoplasm, the staining depth of yellow to brown. The positive rate of NEDD4 L in ovarian cancer was significantly lower than that in ovarian cancer(P<0.05). The positive rate of NEDD4 L in ovarian cancer was significantly lower than that in ovarian cancer.The difference was significant in normal group and benign tumor group(p<0.01).2. The positive expression of NEDD4 L in 41 cases of early-stage ovarian cancer was 25 cases(60.9%), and in 31 cases of advanced ovarian cancer, the positive expression of NEDD4 L was 7 cases(22.5%). The positive expression rate of NEDD4 L decreased with the clinical stage, and the expression of NEDD4 L was significantly different from clinical stage(P<0.01). The positive expression of NEDD4 L protein was 11 cases(61%) in 18 cases of low-grade serous ovarian cancer and 7 cases(30.4%) in 23 cases of high-grade serous ovarian cancer, the difference was statistically significant(P<0.05). The positive expression of NEDD4 L protein in 6 cases(31.5%) was found in 31 cases of mucinous ovarian carcinoma, 19 cases of moderately and poorly differentiated, and 8 cases(66.6%) in 12 cases of highly differentiated. The positive expression rate of NEDD4 L was 14 cases(45.1%). The positive expression rate of NEDD4 L in 41 cases was 18 cases(43.9%). There was no significant difference between NEDD4 L expression and histological type(P>0.05). The positive expression of NEDD4 L was found in 7(22.5%) of 31 cases of ovarian cancer with lymph node metastasis, and 25 cases(60.9%) in 41 cases of ovarian cancer with no lymph node metastasis. The positive expression rate of NEDD4 L was significantly lower than that of lymph node metastasis(P<0.05). There was significant difference between the two groups(P<0.05).3. Kaplan-Meier survival analysis: The expression level of Nedd4 L was closely related to the disease-specific survival rate. The survival time of Nedd4 L high expression group was significantly longer than that of low expression group. The 5-year survival rate of Nedd4L-positive tumors was 59.27%, while the 5-year survival rate of patients with negative expression was 21.32%. Thus, the positive and negative expression of Nedd4 L was significantly different at 5-year survival(P=0.02).4. NEDD4 L was transfected into human ovarian cancer cell line SKOV3, and the expression level of NEDD4 L in the cell line was relatively low. In addition, the stable transfection of NEDD4 L was established by G418 screening. Eukaryotic expression vector and empty vector SKOV3-NEDD4 L and SKOV3-Neo. The expression of NEDD4 L protein in NEDD4L-transfected cell lines could be detected by RT-PCR. The results showed that the expression of NEDD4 L protein in NEDD4 L eukaryotic expression vector was up-regulated in 2200 bp westemblot. In the corresponding empty vector transfected cells and not transfected cells was significantly more.5. To observe the relationship between growth and migration of ovarian cancer cells after transfection of NEDD4 L in vitro. The proliferation rate of SKOV3-NEDD4 L cells was significantly slower than that of SKOV3 and SKOV3-Neo in the control group(P<0.001). The morphological changes of SKOV3 cells after transfection SKOV3-NEDD4 L cells were inhibited by the growth of the cells, such as the cells were round, the volume increased; cytoplasm blue dye, see more vacuoles; 3 nucleoli; 86% of SKOV3-NEDD4 L cells in the nucleus and cytoplasm of SKOV3-Neo and SKOV3 group of cells reduced; heterochromatin condensation, mitochondrial degradation. In the SKOV3-NEDD4 L group, the ultrastructure of the SKOV3-NEDD4 L cells showed apoptotic signs, ie, the cell volume increased, the morphology became uniform, the ratio of nucleus to cytoplasm decreased, vacuolization of cytoplasm, clear nuclear membrane. Nuclear pyknosis, chromatin has a certain degree of aggregation. The SKOV3-Neo group was(131.5±10.2) ?m, the SKOV3-NEDD4 L group was(21.1±7.2) ?m, the SKOV3-Neo group was(21.6±14). The migration distance of NEDD4 L group was significantly shorter than that of SKOV3-Neo and SKOV3 groups(P<0.05). These results show that increased expression of NEDD4 L in ovarian cancer cells can effectively reduce the ability of cell migration and inhibit its growth.6. The effect of NEDD4 L overexpression on cell cycle progression and apoptosis was examined by flow cytometry. SKOV3, SKOV3-Neo and SKOV3 were expressed in SKOV3, SKOV3-Neo and SKOV3 in NEDD4 L cells, NEDD4 L overexpression could induce cell cycle arrest by increasing the number of cells in G2 or S phase. Furthermore, NEDD4 L could significantly increase the number of hypodiploid cells in subphase Gl, suggesting that NEDD4 L could induce Ovarian cancer cell apoptosis.7. Annexin V/P double staining. The results showed that the number of apoptotic cells and the number of advanced apoptotic cells in SKOV3-NEDD4 L cells were increased after treatment with NEDD4 L eukaryotic expression vector by liposome-mediated transfection of SKOV3 cells for 24h(Q4: 5.2±3.3%), And most of the cells were in the quadrant of the quadrant of the pancreas. This indicated that NEDD4 L could induce apoptosis of SKOV3 and the apoptotic cells were mostly in the late apoptotic stage.8. SKOV3, SKOV3-Neo and SKOV3-NEDD4 L cells were inoculated into nude mice respectively. 6 to 7 days after inoculation of SKOV3-Neo, SKOV3 cells in nude mice can be in the armpit to touch the rapid growth of the mass. However, none of the eight mice inoculated with SKOV3-NEDD4 L did not touch the tumor mass within three weeks, and only two of them had small masses after three weeks. The mean tumor volume of SKOV3-NEDD4 L group was significantly lower than that of SKOV3 group and SKOV3-Neo group(0.019±0.005cm3vs0.897±0.207cm3or0.409±0.378cm3, P<0.01). Nude mice were sacrificed in the SKOV3 group and SKOV3-Neo group of nude mice to see larger tumors and visible multiple nodules, but SKOV3-NEDD4 L group of nude mice were not the case. The expression of NEDD4 L in the SKOV3-NEDD4 L group was significantly higher than that in the SKOV3-SKOV3-NEO group(P<0.05). The results showed that the increased expression of NEDD4 L can reduce the tumorigenicity of SKOV3 cells in nude mice, and inhibit tumor growth. Conclusion:1. The expression of NEDD4 L protein in normal ovarian tumor, benign epithelial ovarian tumor, borderline epithelial ovarian tumor, epithelial ovarian cancer tissue decreased gradually, and even the expression of NEDD4 L protein decreased or absent may be associated with epithelial ovarian cancer Occurrence and development. The expression of NEDD4 L protein may be related to the degree of malignancy of epithelial ovarian cancer, and the expression of NEDD4 L protein may be related to the degree of malignancy of epithelial ovarian cancer. Abnormal expression of NEDD4 L protein may be related to the development and invasion and metastasis of epithelial ovarian cancer.2. NEDD4 L can significantly inhibit the proliferation and metastasis of ovarian cancer cells in vitro. The ability of NEDD4 L to induce apoptosis and cell cycle arrest in ovarian cancer cells was related to its inhibitory effect on proliferation. NEDD4 L in nude mice, NEDD4 L can reduce the tumorigenic ability of ovarian cancer cells and extend the incubation period of the tumor. |
PDF Full Text Request