Studies On The Correlation Between Cyclin H And Ovarian Cancer

ObjectiveWe investigated the expression of Cyclin H in human epithelial ovarian cancer and the correlation with human survival and their clinicopathologic significance. Besides, after interfering Cyclin H expression in ovarian cancer cells, we studied the biological behavior changes of cells. To investigate the specific effect of in the occurrence and the development of the ovarian cancer.Methods1. Immunohistochemical technique was used to detect the expression levels of Cyclin H, p-CDK2and Ki-67in60cases of ovarian cancer tissue. To analyze the connection between Cyclin H and clinicopathologic parameters of epithelial ovarian cancer patients, such as age, histological grade, FIGO stage, peritoneal fluidx and so on.60cases of epithelial ovarian cancer patients were followed up, survival curve was calculated using the Kaplan-Meier method.2. Serum starvation and release were used to synchronize ovarian cancer cells. Flow cytometer was used to analyze cell cycle. The expression of Cyclin H and asscioated protein were detected by western blot and nuclear slurry separation technology. We constructed EGFP tagged Cyclin H fusing protein sense expressive plasmid, Cyclin H RNAi recombinant plasmid and EGFP tagged NLS (nuclear localization sequence) absence of Cyclin H fusing protein sense expressive plasmid, transfected into cells, to study the effects of Cyclin H involving in the proliferation and invasion of ovarian cancer cells and its mechanism through MTT assay, Western blot, nuclear slurry separation technology, Transwell and immune precipitation experiments.3. In vivo, nude mice experiments were performed to evaluate the effects of Cyclin H during the growth of ovarian tumors.Results1. Immunohistochemistry analysis showed Cyclin H expression directly correlated with malignant grade of ovarian cancer. Cyclin H positive expression was significantly associated with pathologic stage (Spearman’s r=0.A66; P=0.000), FIGO stage (Spearman’s r=0.389; P=0.002) and if can detect cancer cells from the ascites of the ovarian cancer patients (Spearman’s r=0.308; P=0.016). On other hand, no significant difference were seen regarding patients’ age, histologic subtype, residual tumor and chemotherapy. In all cases of ovarian cancer analyzed, Cyclin H expression was positively associated with p-CDK2(Spearman’s r=0.788; P<0.001) and was positively associated with Ki-67expression (Spearman’s r=0.907; P<0.001). By using the Kaplan-Meier analysis, patients in the high expression Cyclin H (>2+) group were significantly associated with poor overall survival (P=0.003).2. Building the EGFP tagged Cyclin H fusing protein sense expressive plasmid, Cyclin H RNAi recombinant plasmid and EGFP tagged NLS absence of Cyclin H fusing protein sense expressive plasmid, transfected into cells, and then identified cells. MTT assay was detected the proliferation of normal and above stably transfected HO8910ovarian cancer cell lines. Overexpression of Cyclin H promoted the proliferation of HO8910cells (P<0.05). Inhibition of cyclin H expression inhibited the proliferation of HO8910cells (P<0.05). While overexpressed of the NLS absence of Cyclin H fusing protein sense expressive plasmid of HO8910cells, the proliferation of cells hadn’t changed (P>0.05). Advances serum starvation and release of the cells study have revealed:the expressions of Cyclin H, CDK7and MAT1increased, at the same time, the expression of phosphorylation CDK2also increased. Nuclear slurry separation technology showed that Cyclin H, CDK7and MAT1expression were increased in nuclei while no significant changes in the cytoplasm of the proliferative HO8910cells. The coimmunoprecipitation assay displaied that the assembly capacity of Cyclin H, CDK7and MAT1increased in proliferative HO8910cells. Western blot showed overexpressed Cyclin H can found p-CDK2upregulation, while inhibited cyclin H expression can downregulate the levels of p-CDK2in HO8910cells.3. Transwell experiments founded that overexpressed of cyclin H increased the invasive ability of HO8910cells (P<0.05). Vice versa, downexpressed of cyclin H decreased the invasive ability of HO8910cells (P<0.05). And interestingly, overexpressed of the NLS absence of Cyclin H fusing protein sense expressive plasmid of HO8910cells, the invasive ability of cells was significantly enhanced (P<0.05).4. In vitro, interfering Cyclin H expression of ovarian cancer cells can affect ovarian cancer cell tumorigenicity. Overexpressed Cyclin H can promote the growth of tumors, while inhibited cyclin H expression can reduce the growth of tumors.Conclusions1. The expression of Cyclin H was closely correlated with the aggressiveness of ovarian cancer. It indicates that disregulation of Cyclin H may may play key roles in the occurrence and development of ovarian cancer.2. Cyclin H in nuclei of the HO8910ovarian cancer cells may form complexes with CDK7and MAT1, regulating the phosphorylation of CDK2, which may regulate the proliferation of ovarian cancer cells.3. Cyclin H in cytoplasm of HO8910ovarian cancer cells regulated the levels of FAK and phosphorylation Akt, which may regulate the invasion ovarian cancer cells.In a word, these datas revealed that Cyclin H may play a role in the oncogenesis and development of epithelial ovarian cancer and may be a diagnosis or prognostic factor in epithelial ovarian cancer.