Study On The Clinical Significance And Function Of CXXC5 In Epithelial Ovarian Carcinoma

Background and objective:Ovarian cancer (OC) is the fifth lethal cause among gynecologic cancers and is responsible for the deaths of approximately 140,000 women worldwide each year.The survival rate of women with ovarian cancer has changed little more than 20 years ago. Progress in the fight against ovarian cancer has been hampered by lots of factors. These include the difficulty of early detection, the lack of effective treatments for advanced-stage disease and a high degree of molecular heterogeneity in ovarian tumors. The potential pathogenesis of ovarian cancer is poorly understood,CXXC-type zinc finger protein 5 (CXXC5) belongs to CXXC-type family and encodes a nuclear factor, retinoid-inducible nuclear factor (RINF). The CXXC5 gene localizes to the long arm of chromosome 5q31.3 and spans 35.3 kb. Recent advances have indicated that CXXC5 is involved in regulating numerous processes, including cell apoptosis, endothelial cell differentiation, vessel formation and signal transduction.S.Knappskog et al. reported that CXXC5 was overexpressed in several human cancers, including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), primary breast, and thyroid cancers and metastatic melanomas.And high levels of CXXC5 was associated with TP53 wild-type status and might be an unfavorable prognostic factor in breast cancer. However, there has been little research into the influence of CXXC5 on the potential progression and prognosis of ovarian cancer. In addition, the functions of CXXC5 in tumorigenesis and molecular mechanism involved in ovarian cancer are still unknown.Our study is to improve prognosis of patients with ovarian cancer by exploring the relationship between the expression of CXXC5 and clinical characteristic and CXXC5 on the functions of epithelial ovarian cancerMethods:1. GEO and TCGA analysis of CXXC5 in epithelial ovarian cancer We analyzed the alteration of CXXC5 in cancer genomics data of GEO (Gene Expression Omnibus) and TCGA (Cancer Genome Atlas).2. Expression of CXXC5 in ovarian cancer and benign ovarian cyst tissue and its clinical significance in ovarian cancer patientsWe detected the CXXC5 protein in the tissue chips and analyzed the relationship between the expression of the CXXC5 with the clinicopathological features of patients with ovarian cancer by SPSS software.3. The biological functions of CXXC5 in ovarian cancer cells1) We succeeded in constructing ES-2 and COV504 cells with pEZ-Lv105-shCXXC5 stable transfection and OVISE and NIHOVCAR3 with pEZ-Lv05-CXXC5 vector using strategy of lentivirus infection.2) The expression of CXXC5 was confirmed by Western blot and RT-PCR analysis.3) The CCK8 assay was engineered to detect the effect of CXXC5 on cell proliferation.4) The colony formation assay was engineered to detect the effect of CXXC5 on cell proliferation.5) Tumor xenografts in nude mice were further used to certificate proliferative active of CXXC5 in vivo.6) Flow Cytometry was used to examine the cell apoptosis and the cell cycle progression.7) Transwell assay was used to examine the migration and invasion capabilities with ovarian cancer cells.4. Exploring the potential molecular mechanism underlying CXXC5-mediated effects on the ovarian cancer cells proliferation and apoptosis.Western blot method was used to detect the non-phosphorylation and phosphorylation level of Src, Akt, Erk1/2 signaling proteins with CXXC5 overexpression or knockdown.Results:1. CXXC5 is frequently up-regulated in ovarian cancer specimensTo study the discrepancies of CXXC5 in ovarian cancer, immunohistochemical analysis was performed to examine the expression of CXXC5 on 210 paraffin-embedded ovarian cancer tissues. CXXC5 protein was positively detected in 68 in 170 (39.3%) ovarian tissues. There was statistically significant between the expression of CXXC5 in benign ovarian cyst and ovarian carcinoma (P=0.003). Collectively, these findings suggest that CXXC5 is overexpressed in ovarian carcinoma specimens.2. The expression of CXXC5 is closely related to histologic type and lymph node metastasis. Next, we analyzed the relation between the expression of CXXC5 in 170 casesof ovarian cancer specimens and the clinicopathological characteristics of the patients. The CXXC5 expression was not significantly related to patient age (P=0.166) and FIGO stage (P=0.376). Nevertheless, the expression of CXXC5 was significantly correlated with histologic type (P=0.014) and lymph node metastasis (P=0.022). These findings suggested that CXXC5 might be a useful marker for prediciting a histologic type and a poor prognosis and it could play an crucial role in the progression of ovarian cancer.3. CXXC5 promotes ovarian cancer cell growth in vitro and in vivoTo investigate the functions of CXXC5 in ovarian cancer, CCK8 assay and colony formation assay were engineered to detect the effect of CXXC5 on cell proliferation. We found that CXXC5-knowdowning ES-2 cells showed a significantly lower in vitro proliferation than control cells and the proliferation rate of CXXC5-overexpressing OVISE cells was higher significantly than control cells. We further certificated the proliferation in vivo by using tumor xenografts in nude mice. Collectively, these results suggested that CXXC5 could promote ovarian cancer cell proliferation.4. CXXC5 inhibits apoptosis of ovarian cancer cells in vitroFlow Cytometry was used to examine the cell apoptosis and we found that the number of early and late apoptotic cells of CXXC5-knowdowning ES-2 cells was significantly lower than that of control group (shl vs con P=0.0478;sh2 vs con P=0.0243), inversely, the number of early and late apoptotic cells of CXXC5-overexpressing OVISE cells was significantly higher than that of control group (P=0.0448). We conclude that CXXC5 inhibits apoptosis of ovarian cancer cells in vitro.5. Ectopic expression of CXXC5 enhances ovarian cancer cell migration and invasion in vitroNext, to investigate the effects of ectopic expression of CXXC5 on migration and invasion, transwell assay was used to examine the ovarian cancer cells capabilities. In the cell migration and invasion assay, CXXC5-knowdowning ES-2 cells exhibited a significant less of migration and invasiveness, whereas CXXC5-overexpressing OVISE cells showed significantly increase migration and invasiveness. These results suggest that Ectopic expression of CXXC5 enhances ovarian cancer cell migration and invasion in vitro.6. CXXC5 activates the Src, Akt, Erk1/2 pathway in ovarian cancerTo investigate the molecular basis of CXXC5-mediated functions, we first examined the activation of several signaling pathways. The results showed that CXXC5-knowdowning ES-2 could significantly diminish the phosphorylation level of Akt, Erk1/2, Fak and Src. On the contrary, CXXC5-overexpressing OVISE could significantly increase the phosphorylation level of Akt, Erk1/2, Fak and Src. These results confirmed CXXC5 regulated proliferation and apoptosis through Src/Fak, Akt and Erk1/2 signaling in ovarian carcinoma cells.Conclusions:1. CXXC5 might be a marker for prediciting a poor prognosis.2. CXXC5 promotes ovarian cancer cell growth in vitro and in vivo.3. CXXC5 inhibits apoptosis and enhances migration and invasion of ovarian cancer cell in vitro.4. CXXC5 might promote proliferation, migration, invasion and inhibits apoptosis of ovarian cancer cells by activating the Src, Akt, Erk1/2 pathway.