Research Of CIAPIN1in Induced Proliferation Of Epithelial Ovarian Cancer

Epithelial ovarian cancer is the fourth leading cause of ovarian malignantcancer in the world, especially in China. The low survival rate is the first amongGynecology tumor. Despite considerable advances in surgical techniques andneoadjuvant chemotherapy, overall patient outcome has not substantiallyimproved. EOC still remains a clinical challenge mainly due to the absence ofeffective methods for early diagnosis and prognostic mark. Therefore, additionalefforts are needed to find a biomarker for reducing poor outcomes for EOC.CIAPIN1, cytokine-induced apoptotic inhibitor, has been identified to be adownstream effector of the receptor tyrosine kinase–Ras signaling pathway inthe mouse Ba/F3pro-B cell line. It does not show any homology to knownapoptotic regulatory molecules, such as caspase family, Bcl-2family, or signaltransduction molecules, which are proven to be a mediator and play a crucialrole in hematopoiesis. The expression of CIAPIN1was completely dependentedon stimulation with growth factors, such as stem cell factor, interleukin3, andthrombopoietin in factor-dependent hematopoietic cell lines, and forcedexpression of CIAPIN1conferred against apoptosis caused by growth factordeprivation in vitro. However, although CIAPIN1has been observed for6years,the mechanism about the anti-apoptotic molecule function of CIAPIN1is stillnot clear. After following investigation, some investigators found that CIAPIN1 was closely correlated with the progression of many solid tumors andhematological malignancies. Some authors believed that CIAPIN1participatedin multidrug resistance in tumors. Consistently, CIAPIN1was further studied atthe hematopoiesis and was found to decrease in AM-/-erythroid cells, whichinitiated apoptosis during terminal maturation. Thus, CIAPIN1is considered tobe a necessary molecule for hematopoiesis due to the AM-/-deficient mice diein late gestation (between E-12.5and E-14.5) with AM deficient embryos thatare small with fetal livers. Thus CIAPIN1was not only regulated cellularapoptosis in vitro but also participated in cellular differentiation and organicmaturate in vivo. Together, it is implied that CIAPIN1might affect thedevelopment of EOC. CIAPIN1may be a promising biological target fortreatment of EOC.About over90%EOC derived from ovarian epithelial cells, whichdeveloped the function of potential multi-differentiation. The exact mechanismof EOC is still unknown. Herein, we want to explore the basic biologicalfunction of CIAPIN1in the proliferation of EOC, which could help usunderstand the progression of EOC.ObjectiveTo investigate the expression of CIAPIN1in EOC tissues and investigatethe biological function of CIAPIN1in EOC cell lines, discuss the mechanism ofCIAPIN1on EOC.Methods1. The expression and significant of CIAPIN1in118EOC tissues and108non-cancerous tissues were detected by immunohistochemical assay, thenthe difference to FIGO stage, pathological subtype, and histological differentiated and other clinical parameters were evaluated.2. To verify the result of immunohistochemical assay, the western bolt assaywas used to detect the diference of CIAPIN1protein in8paired EOCtissues and non-cancerous epithelial tissues.3. To certify CIAPIN1expression in EOC tissues, the difference of CIAPIN1expression in EOC cells were detected by western blot、immunohistochemical and immunofluorescence4. Knockdown and overexpression of CIAPIN1in EOC cells were detected byreal time quantity PCR and western blot.5. The reproductive activity was detected by MTT、Soft agar assay and cellcycle analysis in A2780CIAPIN1siRNA cells and controls. Tumorigenesisassay confirmed those effects in vivo.6. The reproductive activity was detected by MTT、Soft agar assay and cellcycle analysis in HO8910AdCIAPIN1cells and controls. Tumorigenesisassay confirmed those effects in vivo.7. Western blot was used to detect the relationship between CIAPIN1and cellcycle proteins as well as NF-κB proteins.8. Immunofluorescence was used to detect the translocation of NF-kB protein.Reults1. CIAPIN1expression in tumors was notable higher than that in pairednon-cancerous epithelial tissues. Further analysis revealed that CIAPIN1staining in poorly and moderately differentiated tumor tissues weresignificantly higher than that in well differentiated tissues, and clearly higherin advanced FIGO stage (III+IV) than that in early stage (I+II) patients.No signi cant relationship was observed between age, lymphatic metastasis and pathology subtypes.2. High expression of CIAPIN1was verified in five EOC cell lines (HO8910,OVCAR3, A2780,3AO, SKOV3). The significant significance of CIAPIN1expression was obtained in A2780and HO8910.3. The difference of A2780and HO8910was verified by immunohistochemicaland immunofluorescence.4. The recombinant CIAPIN1siRNA was constructed successfully.5. The MTT assays demonstrated that the growth rate of A2780cells wassignificantly lower in CIAPIN1siRNA cells compared with that in twocontrols. The colony number (foci>50μm) of down-regulating CIAPIN1cells was less than in other controls after15days’ culture, indicating thatdown-regulation of CIAPIN1could decrease tumor growth potential of EOCcells. The flow cytometry analysis showed that the CIAPIN1siRNA cellshad markedly higher in G1stage and lower in S stage compared withcontrols. This result demonstrated that the CIAPIN1siRNA cells hadmarkedly lower in G1stage and higher in S stage compared with controlsrespectively. But all the indexes had no significant difference betweennegative and blank controls. The findings indicated that the proliferation ofA2780cells infected with adenovirus-mediated CIAPIN1siRNA wasmarkedly arrested in the G1stages. Tumorigenesis assay confirmed thoseeffects in vivo.6. The mRNA and protein of CIAPIN1were significantly elevated inAdCIAPIN1cells compared with those in two controls. The MTT assayconfirmed that AdCIAPIN1cells presented much faster growth compared tothe controls, and statistical analysis showed a significant difference. After15-day culture, the colony number of AdCIAPIN1was more than those of the controls. The flow cytometry analysis showed that the AdCIAPIN1cellshad markedly lower in G1stage and higher in S stag compared with controls.These results suggested that ectopic CIAPIN1expression could enhanceEOC cell proliferation and promote cell growth in vitro. Tumorigenesisassay confirmed those effects in vivo.7. CIAPIN1could upregulate cell cycle proteins cyclinD1, CDK4, CDK2andcyclinE, and active NF-κB pathway.8. NF-κB/P65was observed in HO8910AdCIAPIN1nucleus, but norelationship between Control and AdEGFP controls by western blot andhigh-content NF-κB translocation assay on ArrayScan HCS cytometer. Theimmunofluorescence result showed that NF-κB/p65was not activated inA2780Controls、Con-siRNA and CIAPIN1siRNA.ConclusionCIAPIN1might promote EOC cell growth and proliferation byupregulating cell cycle proteins and activating transcription factor NF-κB whichexecutes its function by performing nucleus translocation.