The Function Of Human Phosphatidylethanolamine-Binding Protein In The Apoptosis Induced By TRAIL

Ovarian cancer is a kind of serious gynecological malignancy,which can severely impire the women's health.It is estimated that as many as 23,000 new cases and 14,000 deaths from ovarian cancer in the United States each year.Currently,ovarian cancer is the first leading cause of gynecological cancer-related deaths in China.But the early symptoms of ovarian cancer is obscure and secretly,and is often diagnosed only after the disease has reached an advanced stage. The advanced stage of ovarian cancer is characterized by metastasis outside of the pelvic region to the peritoneum or even to tissues beyond the peritoneum, which makes the treatment difficult. Most women with ovarian cancer present with advanced disease and are treated with cytoreductive surgery followed by platinum-based chemotherapy, Although these women initially respond to treatment, the majority will relapse and eventually succumb to resistant disease.So it is imperious task to overcome multidrug resistance and look for new strategies to combat the disease for tumor therpy.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL),also known as Apo2 ligand is a new member of the tumor necrosis factor superfamily of cell death-inducing Hgands.Similar to other members of the family,TRAIL can mediate the death signals and trigger apoptosis through death receptors on the membrane of cells.But the most remarkable features of TRAIL is its ability to induce programmed cell death or apoptosis in a variety of tumor cell lines but not typically in normal or nontransformed cells.At present, approximately 50% of malignant tumors are sensitive to TRAIL. These features make TRAIL a desirable therapeutic agent to fight cancer.Phosphatidylethanolamine-binding proteins (PEBPs) is an evolutionary conserved protein family that was originally identified from bovine brain and shows preferential affinity in vitro of phosphatidylethanolamine, a component of the cell membrane.It has a molecular weight of 21-23kDa and can be found in species of flowering plants , parasites, nematodes, insects, and mammals, including cows, monkeys and humans.Researches suggest that the function of PEBP in mammals islipid binding, inhibition of serine proteases ,however,the specific physiological role of PEBP is not clear. Up to date, four kind of human PEBP have been found,one of which is hPEBPl ,as a suppressor of Raf-1 kinase activity and mitogen-activated protein (MAP) kinase signaling.also called Raf kinase inhibitory protein (RKIP).hPEBP4 ,as a new member of PEBP family,has been purified from human bone marrow stromal cells (BMSC) by the researchers of institute of immunology in Second Military Medical University. Homology analysis revealed close similarity (about 30-60%) to other known members of PEBP family, such as human, mouse, bovine, rat, Drosophila melanogaster and Oryctolagus cuniculus PEBPs and PEBP-like proteins.The previous work of this institute of immunology shows that mRNA expressions of hPEBP4 appear strong message in MCF-7(breast carcinoma), PC-3 (prostate carcinoma) and Caov-3 (ovarian carcinoma) cells and hPEBP4 as a antiapoptosis molecules play an important role in TNFa-induced apoptosis.Primary evidence show Caov-3 with high mRNA expression of hPEBP4 is sensitive to TRAIL a new member of TNF family.To us interesting,is there any relationship between hPEBP4 and TRAIL? In the following researches,we will investigate wheather hPEBP4 can contribute to the apoptosis induced by TRAIL in ovarian cancer Caov-3 and explore related signal mechanism.Part I Construction of hPEBP4 RNA interference expressing vector,selection and identification of infectious cellsObjective:To construct the hPEBP4 RNA interference expressing vector with RNA interference technology, then to stably select the infectious Caov-3 cells with the vector and the selected cells eventually are identified.MethodsrAccording to the general law for design of primer and the feature of restriction endonuclease(RE) sites in RNA interference expressing vector pSuppressorNeo,two piece of RNA interference primers were synthesized.The empty vector pSuppressorNeo was digested by restriction endonuclease Sal I and Xba I ,then primers were inserted between the two RE site in the vector. After ligation,the plasmid was transformed DH5 a and positive clones were appraised correct by sequencing.Then RNA interference plasmids were infected into Caov-3and selected resistant clones by G418,which were identified by RT-PCR and Western blot at last.ResultsrConstructive plasmids were identified preliminary by PCR and RE digestion and sequencing correct ultimately.Caov-3 cells were infected by the RNAi plasmids and through stable selection,the expression of hPEBP4 completely disappeared ,which were proved by RT-PCR and Western blot.The control and unrelated interference plasmid had no effect on the expression of hPEBP4.Conclusion: The successfulconstruction of RNA interference expressing vector inhibited the expression of hPEBP4 efficiently and specifitly . RNA interference technology provide us a powerful tools to explore functions of genes.Part II The function of hPEBP4 in the apoptosis induced by TRAILObjective:To observe the difference in apoptosis and signal transduction between Caov-3 with and without RNA interference to the treatment of TRAIL, and estimated the possible function of hPEBP4 in the induced apoptosis by TRAIL.Methods: The condition of cells was visualized morphologically by phase contrast microscopy.RT-PCR analysis confirmed that the expression of hPEBP4 were regulated and controlled by TRAIL.Apoptosis induced by TRAIL was examined by flow cytometry and signal transduction was detected by Western blot.Results: In quiescent condition,no significant difference in apoptosis was observed between cells with RNAi or not. But some time after treatment with TRAIL, changes of apoptosis in morphology were visualized obviously,and Caov-3 without RNAi or unrelated RNAi showed much more apoptosis than ones with hPEBP4 RNAi,in which the rate of apoptosis is much lower by FACS analysis.The evidences from Western blot showed the activation of ERK and MEK had been increased in Caov-3 with RNAi,however,in parent Caov-3 cells apoptosis was in company with phosphorylation of JNK and p38.Increment of apoptosis was obtained when MEK7ERK signal pathway was inhibited by PD98059 pretreatment.Conclusion: The expression of hPEBP4 could be regulated by TRAIL.Caov-3 cells with RNA interference was relatively resistant to the apoptosis induced by...