The Effect And Mechanism Of Trim27on Growth Of Ovarrian Cancer Cells The Expression Of A Novel Anti-Inflammatory Cytokine IL-35and Its Possible Significance In Childhood Asthma

Objective:Ovarian cancer is one of the common malignant tumors of female gential organs, and its incidence is just second to cervical and uterine cancer. However, its mortality accounts for the fisrt place in all kinds of gynecological cancer. Thus, ovarian cancer is still a serious threat to female. More than90%of ovarian cancers are epithelial ovarian cancer, and cystadenocarcinoma is the most common type of epithelial ovarian cancer. Surgery combined with chemotherapy is the commonly clinical treatment for ovarian cancer, however, it is hard to detect in early stage and women with ovarian cancer may have no symptoms or just mild symptoms until the disease is in an advanced stage, for which the5-year survival rate is still less than30%. Now the etiology and molecular mechanisms of ovarian cancer are still not well known. Therefore, it is very necessary to explore the pathogenesis of ovarian cancer at the molecular level, and develop molecular diagnostics marker for effective treatment to ovarian cancer.TRIM27was originally identified as the fusion partner with the RET proto-oncogene. TRIM27is also known as RFP (ret finger protein) and belongs to the zinc finger protein superfamily. TRIM27contains a tripartite motif consisting of a RING finger, B-box zinc finger and coiled-coil domain. TRIM27becomes oncogenic when its tripartite domain is recombined with the tyrosine kinase domain of the RET proto-oncogene by DNA rearrangement, and displays tumorigenic activity in mouse embryos NIH3TS cell. It has been reported that TRIM27is highly expressed in endometrial cancer, breast cancer and lung cancer, and correlated with a poor prognosis of the patients, suggesting that it may be involved in tumorigenesis and development. In our study, we aim to study the expression levels of TRIM27in cystadenocarcinoma and normol ovarian epithelial tissues by immunohistochemistry, and analyze the correlation between TRIM27expression in cystadenocarcinoma tissues and clinicopathological parameters. We try to explore the roles of TRIM27downregulation in proliferation, colony forming ability, cell cycle and apoptosis of ovarian cancer cells by specific TRIM27siRNA, and clarify the effective mechanism of TRIM27in ovarian cancer.Materials and Methods1. Clinical dataSamples were taken from Jinan Central Hospital affiliated to Shandong University from2001to2012, including50cases of cystadenocarcinoma patients aged30to79years old, the average age is50, None of them had received radiotherapy, chemotherapy prior to surgery and they all have complete clinicopathological data. We also collected16normal ovarian tissues from the non-ovarian disease patients who were underwent surgical removal of the ovaries. The pathological diagnosis was made according to the current World Health Organization (WHO) criteria, clinical staging was according to the Figo system.2. The expression of TRIM27protein in cystadenocarcinoma specimens and normal ovarian tissues detected by immunohistochemical (IHC)TRIM27expression levels in50cystadenocarcinoma and16normal controls were detected by IHC, and the relationship between TRIM27expression and clinicopathological parameters was examined by the Chi-square test.3.The effect of decreased TRIM27expression on the prpliferation, cell cycle and apoptosis of ovarian cancer cells(1) We firstly detected TRIM27mRNA and protein expression in ovarian cancer cells including SKOV3, OVCAR3, CAOV3and3AO by RT-PCR, q-RT-PCR and western blot respectively.(2) We chose ovarian cancer cell lines SKOV3and OVCAR3for transfection of TRIM27siRNA or negative control respectively by liposome mediated method. The efficiency of knockdown was identified by RT-PCR and western blot.(3) Cell viability was evaluated by CCK8assay in SKOV3and OVCAR3cells after TRIM27downregulation.(4) Colony formation assay was used to determine cell colony formation capacity of SKOV3and OVCAR3cells after TRIM27downregulation.(5) The cell cycle of SKOV3and OVCAR3cells was detected by flow cytometry after TRIM27downregulation.(6) The apoptosis ratio of SKOV3and OVCAR3cells was detected by flow cytometry after TRIM27downregulation.4. The effect of reduced TRIM27expression on the cell proliferation-related pathways.(1) The effect of TRIM27downregulation on MAPK pathway in SKOV3and OVCAR3cells was detected by western bot.(2) The effect of TRIM27downregulation on AKT pathway in SKOV3and OVCAR3cells was detected by western bot.Results1.The different expression and clinical significance of TRIM27in cystadenocarcinoma and normal ovarian tissues(1) TRIM27expression levels is upregulated in cystadenocarcinoma tissuesIHC results from cystadenocarcinoma tissues showed that about18%(9/50) patients was negative,20%(10/50) expressed low level of TRIM27,44%(22/50) expressed medium level of TRIM27, and18%(9/50) expressed high level of TRIM27. While in normal controls,37.5%(6/16) was negative,37.5%(6/16) expressed low level of TRIM27, only2patients expressed medium level of TRIM27and2patients expressed high level of TRIM27. By statistical analysis, the expression of TRIM27protein in cystadenocarcinoma was significantly higher than that in normal controls (p=0.0098).(2) High expression of TRIM27has a positive correlation with Figo stage in cystadenocarcinoma patientsWe further analyzed the relationship between the TRIM27expression and clinical pathological parameters in cystadenocarcinoma patients. The results showed that there was a significantly positive correlation between TRIM27expression and Figo stage in cystadenocarcinoma patients. Figo stage was related to tumor size and metastasis, which suggested that TRIM27may involve in tumor growth and migration. There were no significant correlations among TRIM27expression and age, gender, origin parts and pathological grade.2. The downregulation of TRIM27expression in ovarian cancer cells can suppress cell proliferation and cell cycle, and promote apopotosis. (1) TRIM27is highly expressed in some of human ovarian cancer cell linesThe expression status of TRIM27in human serous cystadenocarcinoma-derived SKOV3, CAOV3, OVCAR3and human mucous cystadenocarcinoma-derived3AO cells was detected by RT-PCR and western blot. The results showed that TRIM27was expressed in these four cell lines, but the expression of TRIM27in3AO cell line was very low. The data of real-time PCR showed that TRIM27expression was high in SKOV3, moderate in OVCAR3cells and low in CAOV3cells.(2) Downregulated expression of TRIM27inhibited the proliferation of ovarian cancer cellsWe chose SKOV3and OVCAR3cells for transfecting TRIM27siRNA or negative control, and the results of RT-PCR and western blot demonstrated the interference efficiency was about50%. CCK8assay was used to detect cell viability in SKOV3and OVCAR3cells. The result showed that the downregulation of TRIM27expression significantly inhibited the proliferation of SKOV3and OVCAR3cells.(3) Decreased TRIM27expression prevented colony formation in ovarian cancer cellsSKOV3and OVCAR3cells were used in colony formation assay, the results indicated that the cloning efficiency and clone size in siTRIM27group were significantly reduced compared with sicontrol group and untreated group (p<0.05).(4) Reduced TRIM27expression inhibited the cell cycle of ovarian cancer cellsCell cycle of SKOV3cells was detected by flow cytometry and the results demonstrated that cell number of S phase in siTRIM27group was markedly increased compared with sicontrol group and untreated group (p<0.05).(5) The decrease of TRIM27expression promoted the apoptosis of ovarian cancer cellsEarly apoptosis was detected by Annexin V/PI double dyeing. We found that reduced TRIM27expression in SKOV3cells resulted in significantly higher apoptosis (p<0.05).3.TRIM27played important roles by affecting cell proliferation-related pathwaysMAPK pathway and AKT pathway were analyzed by western blot. The results showed that downregulated TRIM27led to increased expression of p-P38in SKOV3and OVCAR3cells, while the expression of p-ERK1/2and p-SAPK/JNK did not changed. In addition, downregulated TRIM27also suppressed the expression of p-AKT.Conclusion1.TRIM27expression was significantly increased in cystadenocarcinoma tissues compared with normal ovarian epithelial tissues, and there was a positive correlation between TRIM27protein expression and Figo stage, which suggesting that TRIM27may be related to tumor growth and metastasis.2. The downregulation of TRIM27expression could inhibit, the proliferation, colony forming ability, cell cycle of ovarian cancer cells and promote the apoptosis of ovarian cancer cells.3.Decreased TRIM27expression could promote the expression of p-P38and inhibited the expression of p-AKT, suggesting that TRIM27may play important roles by affecting cell proliferation-related signaling pathway.OriginalityTRIM27, which is acted as the fusion partner with the RET proto-oncogene, is closely related with tumor, however, its effective mechanism in tumor is still not clear. In our study, we reported for the first time that TRIM27is highly expressed in cystadenocarcinoma compared with normal ovarian epithelials tissues, and TRIM27expression was significantly correlated with Figo stage. TRIM27can promote activation of AKT pathway while inhibit the p-P38activation, induce the changes of cell cycle and apoptosis, and then promote proliferation of ovarian cancer cell and colony forming ability of tumor cells. The above study may provide a foundation for exploring the effective mechanism of TRIM27in ovarian cancer, and provide a new target for diagnosis and treatment of ovarian cancer. Objective:Childhood asthma is a chronic inflammatory disease of the airways in children and has become increasingly prevalent in the world. Childhood asthma has become a serious global health problem that is of great concern among medical and health professionals. Childhood asthma is induced by bronchial hyper-responsiveness to specific allergen, and it is mainly characterized by the infiltration of mast cells, basophils, eosinophils, mononuclear cells and the increase of serum total immunoglobulin E (IgE). The imbalance between Thl and Th2responses plays a pivotal role in the pathogenesis of childhood asthma. Excess Th2response increased the eosinophilia and IgE production, then induced airway hyperresponsiveness and chronic inflammation.Interleukin-35(IL-35) is a novel anti-inflammatory cytokine and has been shown to play an important role in maintaining immune homeostasis. It has been reported that IL-35effectively attenuated the inflammatory response in established mice model with collagen-induced arthritis, accompanied by suppression of IL-17production but enhanced IFN-γ synthesis. However, the effect of IL-35on human asthma remains unclear. The present study is to investigate the expression and significance of IL-35in childhood asthma.Materials and Methods1. Collection of the samplesForty-one asthmatic children and forty-two healthy controls were recruited in Qilu Children’s Hospital of Shandong University between March2011to November2013. The diagnosis of asthma was made according to the criteria for childhood asthma established by Chinese Society of Respiratory Diseases.2. Detection of serum total IgE level and eosinophil countSerum total immunoglobulin E level was measured by radioimmunosorbent test. Peripheral blood eosinophils were counted using BC-5800Automatic Blood Cell Analyzer.3. Detection of IL-35mRNA in peripheral blood mononuclear cells from asthmatic children and healthy controls.Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation from peripheral blood of childhood asthma patients and healthy controls. Total mRNA was isolated using a modified TRIzol one step extraction method. cDNA was synthesized using the Rever Tra Ace qPCR RT Kit according to the manufacturer’s instructions. Quantitative real-time polymerase chain reaction for P35, EBI3, P28, P40and GAPDH was respectively performed.4. Measurement of serum IL-35, IL-4and IFN-γ levels by ELISASerum IL-35levels were determined using enzyme-linked immunosorbent assay (ELISA) kits (Cusabio Biotech, Wuhan, China), serum IL-4and IFN-γ levels were also determined using ELISA kits (eBioscience, San diego, USA) according to the manufacturer’s instructions. The correlations among the above indexes were also analyzed using Pearson’s method.Results1. Serum IgE levels and eosinophil count in asthmatic children and healthy controlsFirstly, we detected serum total IgE levels and peripheral blood eosinophils in asthmatic children and healthy controls. Our results showed that serum total IgE levels were significantly higher in asthmatic patients than those in healthy controls and peripheral blood eosinophils were also significantly elevated in the children with asthma compared with healthy children. We further analyzed the relationship of serum IgE levels and eosinophil count in all asthmatic patients, the result showed there was no statistically significant positive correlation between serum IgE levels and eosinophil count2. Serum IL-4and IFN-y levels in asthmatic children and healthy controlsWe detected serum IL-4and IFN-γ levels in asthmatic children and healthy controls by ELISA. The levels of serum IL-4were significantly increased in asthmatic children compared with healthy controls (p<0.001), but serum IFN-γ levels in asthmatic patients were significantly lower than those in normal controls (p<0.001). We also found that there was an obviously positive correlation between serum IgE and IL-4levels in asthmatic patients (r=0.6009, p<0.001). In addition, significantly negative correlation was found between serum total IgE and IFN-γ levels.3. IL-35mRNA expression in PBMC from asthmatic children and healthy controlsWe detected IL-35mRNA expression in PBMC from asthmatic children and healthy controls using qRT-PCR. The results showed that the expression of IL-35subunits (P35and EBI3) mRNA was significantly down-regulated in PBMC from asthmatic patients compared with normal controls (p<0.001for P35and p<0.05for EBI3)4. Serum IL-35levels in asthmatic children and healthy controlsWe detected the expression of IL-35protein in the serum from patients and controls using ELISA. The results demonstrated that serum IL-35levels in asthmatic patients were significantly lower than those in healthy controls (p<0.05). In addition, serum IL-35levels were significantly inversely related to IL-4levels (r=-0.3520, p=0.0327), and significantly positive correlation was also found between serum IL-35and IFN-γ levels (r=0.3569, p=0.0220). However, there were no significantly negative associations of serum IL-35with IgE (r=-0.2909, p=0.0807) and eosinophil count (r=-0.1206, p=0.4586).Innovation and significanceWe report for the fist time that asthma children have decreased expression of IL-35in PBMC and serum. And serum IL-35levels are significantly inversely related to serum IL-4levels, moreover, IL-35level was positively related to serum IFN-γ level. We try to reveal the potential role of IL-35in the pathogenesis of asthma. This will be helpful for looking for a new target for the treatment of asthma.