Function And Mechanism Studys Of CFTR In Tumor Invasion And Metastasis Of Epithelial Ovarian Cancer

Ovarian cancer is one of the most common malignancy in the female reproductive system, with epithelial ovarian cancer being the most common pathological type. In developed countries, it is the most lethal gynecologic cancer since>70% of women were diagnosed with advanced stage and cure rates at this stage are <30%. Identification of molecular markers or pathways may be useful in determining the potential therapeutic targets or novel therapeutic methods.The cystic fibrosis transmembrane conductance regulator (CFTR), an-170 kDa glycosylated protein, is known as a cAMP-dependent chloride (C1-) anion conducting channel. CFTR is found in epithelial cells of human tissues, including the female reproductive tract. By controlling ion and protein transport, CFTR is thought to function in most human cells to assist in the maintenance of cell homeostasis. CFTR belongs to the ATP-binding cassette (ABC) transporter family, members of which utilize ATPase activity to transport substrates across cell membranes and which are involved in various types of cancer. The CFTR gene mutation may lead to dysfunction at the plasma membrane. Such mutations were associated with an increased or decreased risk of cancers. A study proposed that CFTR is associated with epithelial-to-mesenchymal transition in breast cancer. CFTR is also known to be involved in modulating signaling pathways in cell inflammation and apoptosis. Although previous studies suggested a role for CFTR in various types of cancer, the relationship between CFTR and ovarian cancer remains to be determined. Therefore, in the present study, we aimed to identify the potential role of CFTR in the tumor aggression behaviors in epithelial ovarian cancer by the following three parts.PART ONE THE EXPRESSION AND CLINICOPATHOLOGICAL ASSOCIATION OF CFTR EXPRESSION IN HUMAN EPITHELIAL OVARIAN CANCERObjectives:The study was aimed to identify the expression and association of CFTR expression with clinicopathological characteristics in human epithelial ovarian cancer.Methods:(1) Immunohistochemical staining analysis was performed to detect the expression of CFTR in 83 cases of human epithelial ovarian cancers pecimens,18 benign epithelial tumor and 11 normal ovarian tissues.(2) The association of CFTR protein expression with clinicopathological characteristics in cases of human epithelial ovarian cancer was investigated.Results:(1) The expression of CFTR in ovarian cancer specimens was significantly higher than that in benign (P<0.05) and normal ovaries (P<0.05).(2) Regarding the clinicopathological characteristics, the statistical analysis revealed that the CFTR protein level was well-related to advanced clinical stages (stage Ⅲ/Ⅳ vs. Ⅰ/Ⅱ, P<0.05), poor histological grade (grade 2-3 vs.1, p<0.05), and a higher serum Ca-125 level (P<0.05). Furthermore, we observed that CFTR staining was stronger in the serous type as compared to the mucinous(P<0.05) and endometrioid type (P<0.05).Conclusions:Our present study finds that CFTR is overexpression in human epithelial ovarian cancer tissues which associated with an unfavorable clinical prognosis. The expression level of CFTR is higher in serous ovarian carcinoma than other pathological types, which may plays an important role in malignant behavior of serous ovarian carcinoma.PART TWO SILENCED OF CFTR EXPRESSION BY CFTR-SPECIFIC shRNA EXPRESSION pGFP-V-RS WITH THE STABLY TRANSFECTED CELL LINES AND CONATRUCT THE RECOMBINANT ADENOVIRUS OF Ad-CFTRObjectives:The study was aimed to silence CFTR expression by targeting human CFTR gene shRNA expression vector and selected the stably transfected cell lines by puromycin screening. And we aimed to constructe the recombinant adenovirus of Ad-CFTR which contain human CFTR gene. The further research could be performed with this stable transfection of ovarian cancer cell lines and the recombinant adenovirus.Methods:Amplification CFTR-specific shRNA vector and identification by enzyme digestion experiment. The cells were transfected with plasmid by PolyJet TM into A2780, SKOV3 cells. The stably transfected cells were selected by puromycin screening and fluorescence microscopy. Strong green fluorescence was detected under fluorescence microscopy. Western blot was to detect CFTR protein level of CFTRi group, negative control group and not transfection control group respectively. And the CFTRi cells which CFTR protein level was significantly reduced compared with that of its corresponding control was selected.Human CFTR gene was digested, separated and purified. CFTR gene was directional cloned into the shuttle plasmid pAdTrace-TOX vector. Construction of recombinant adenoviral plasmids by homologous recombination in E.coli BJ5183 cells. Transfection of recombinant adenovirus into HEK293 cells to package the recombinant adenovirus. Western blot was performed to detect the expression of CFTR in ovarian cancer cells after virus infection.Results:CFTR-specific shRNA vector was amplified successfully and identified by enzyme digestion experiment. Concentration of puromycin screening was 900 ng/mL for A2780 and 500 ng/mL for SKOV3 cells, respectively. The CFTR-shRNA-1, CFTR-shRNA-2, CFTR-shRNA-3, CFTR-shRNA-4 and negative control vectors were transfected into A2780 and SKOV3 cells. The stably transfected clones were selected by puromycin screening. Clones with GFP expression were detected successfully under fluorescence microscopy. Western blot results revealed that the CFTR protein level of CFTRi cells was significantly reduced compared with that of its corresponding control cells. Thus, SKOV3-CFTRi3 and A2780-CFTRi2 were used in the subsequent experiments.The products of digested by enzymes showed that human CFTR gene was separated successfully after electrophoresed on agarose gel. The plasmid pAdTrace-CFTR-TOX was verified by electrophoresed on agarose gel and Sequencing. The homologous recombination plasmid pAdTrace-CFTR was verified by electrophoresed on agarose gel and PCR. We constructed the adenovirus plasmid Ad-CFTR successfully by RFP reperssion of HEK293 cells under fluorescent microscopy. Western blot analysis showed that Ad-CFTR could effectively infect ovarian cells and increase the protein expression of CFTR.Conclusions:CFTR-specific shRNA vector was amplified successfully and identified by enzyme digestion experiment. CFTR expression was silenced by targeting human CFTR gene shRNA expression vector and the stably transfected cell lines were selected by puromycin screening. We constructed recombinant adenovirus of Ad-CFTR successfully with pAdEasy system successfully. We completed the basic work for the next investigationPART THREE CFTR INVOLVED IN REGULATION OF MIGRATION AND INVASION BEHAVIOR OF EPITHELIAL OVARIAN CANCER CELLS AND THE POTENTIAL MECHANISMSObjectives:This study was aimed to investigate the effect of CFTR on the behavior of invasion and metastasis and to elucidate the underlying mechanisms of CFTR-induced cell motility and invasion of epithelial ovarian cancer.Methods:Transwell assay was used to analyze the ability of CFTRi cell in migration and invasion. Cell migration was evaluated by scratched wound healing assay in CFTR overexpression cells. CFTRi cell was tested by cell adhesion assay. CFTRi cell was detected by CCK-8 assay. CFTRi cell was evaluated by colony formation in vitro. The tumor formation of CFTRi cells was assessed by xenograft tumor formation. Protein expression of Src kinase and phospho-Src which regulated cancer cell metastasis was evaluated in Ad-CFTR infected and contorl cells by western blot.Results:(1) Knockdown of CFTR inhibits cell motility in vitro.For SKOV3-CFTRi and A2780-CFTRi, an ～41.72 and 63.11% reduction in the number of migratory cells was observed compared with SKOV3-NC and A2780-NC cells (P<0.05 for both)(2) CFTRi cells showed a decreased cell invasion in vitro.SKOV3-CFTRi3 cells exhibited an impaired invasion capacity of 60.89% compared to SKOV3-NC cells. Similarly, A2780-CFTRi2 cells had a reduced invasion capacity of 63.93% compared to A2780-NC cells (P<0.05).(3) CFTR-overexpression cells showed a increased cell motility in vitroA2780-Ad-CFTR cells exhibited an increased motility capacity of 39.5%.Similarly, SKOV3-Ad-CFTR cells had a increased motility capacity of 39.3% compared (P<O.05).(4) Knockdown of CFTR inhibits cell adhesion in vitro.There was a 42.31 and 38.52% decreased percentage of optical density (OD) value in SKOV 3-CFTRi and A2780-CFTRi cells compared to the control cells (P<0.05 for both).(5) Knockdown of CFTR inhibits proliferation and in vitro.The results of the CCK-8 assay showed that the growth ability of SKOV3-CFTRi3 and A2780-CFTRi2 cells decreased compared with the corresponding control cells.(6) Knockdown of CFTR inhibits colony formation in vitro.CFTR knockdown cells also had a low colony formation ability as the number of colonies of CFTRi cells were decreased when compared with the corresponding control cells.(7) CFTR knockdown inhibits xenograft tumor formation.We observed that the tumor size of the SKOV3-CFTRi3 group was statistically significantly smaller than that of the SKOV 3-NC group.The size of xenograft tumors of SKOV3-CFTRi was significantly smaller than that of the control. At the same time, the average tumor weight of SKOV3-CFTRi3 group was lower than control group.(8) Our findings indicated that the level of total Src expression and phospho-Src (Tyr419) in every group was no statistical significance. However, Ad-CFTR cells showed the lowest activity of phospho-Src (Tyr530).Conclusions:CFTRi cells showed the lower abilities of cell migration, invasion, adhesion, colony formation and cell proliferation in vitro. Ad-CFTR infected cells showed the increase of the ability to migrate. CFTR may be affected phosphorylation of Tyr-527/530 through the interaction of EBP50, Cbp and Csk indirectly, rather than Tyr-416/419. It might be easier to induce progression of ovarian cancer if be a relatively high expression of CFTR, including migration and invasiveness by activation of c-src signaling pathway.