Screening Of Differentially Expressed Proteins In Epithelial Ovarian Carcinoma With Pathway Array Technique And Research Of Signaling Pathway

Epithelial ovarian cancer is the third common cancer in female gynecological malignancies, but the mortality rate of EOC is the first one among the female reproductive tract malignant tumors. The ovarian locates deep in the pelvis, there were not typical early symptoms of the disease or even no obvious symptoms, that is why early diagnosis rate of EOC is not high. Only about 20% patients were diagnosed in stage I, the other 2/3 of patients were diagnosed at stage III or IV. In the recent years, although rapid progress had been made in surgery and chemotherapy following surgery, the advanced EOC patients had poor prognosis, 5-year survival rate was only about 20%, while survival rate of stage I EOC was up to 80-90%. Therefore, the study of ovarian cancer pathogenesis, markers of early diagnosis and treatment methods is of great significance.The progress of proteomics helps to provide a theoretical basis and solution to clarify the mechanism of disease and overcome the disease. By comparative proteomics research, the detect of differences from sick individual to the normal individuals, "specific disease-related proteins" could be found out, which can not only serve as markers for early diagnosis of disease, but also can be a molecular target for new drugs. Pathway Array technology systems choose some high-affinity antibodies to analyze over a thousand of proteins in a single sample, including the activated phosphorylated proteins and non-phosphorylated total proteins, to help to find signal transduction molecules changes in more comprehensive way. In Pathway Array technology, protein samples could be extracted from cell lines cultured in vitro and ultra-low temperature frozen tissue samples, then the proteins were separated by SDS-PAGE electrophoresis, transferred to NC membrane and then fixed the NC membrane in a Western blotting manifold that isolates 20 channels across the membrane, then add 600 ul of primary antibodies (diluted in blocking buffer) to each channel, each channel contains two to four antibodies, the molecular weight are significantly different from each other in the same channel, which benefits the access to the image. After hybridization, the NC membranes are analysed by Quantity One software Chemi XRS system to obtain images of different protein bands by chemiluminescence technology reflects the combination of the antibodies by specific proteins, and then by Chemi Doc system to make sure protein molecular weight corresponding to each phosphorylation proteins band or non-phosphorylated proteins band for protein characterization, and get relative expression volume of proteins by analysis of expression volume of each protein compared to the internal reference for quantitative comparison. NC membrane with the antibodies were cleaned by stripping buffer, and then another set of antibodies could be blotted. Phosphorylation antibodies are blotted first in order and then the non-phosphorylated antibodies were blotted. Blotting of non-phosphorylated antibodies can be repeated several times, 300-400 antibodies could be blotted in a single film. The advantages of pathway array technology include:①With pathway array technology, there are more specificity and accuracy in identifying the correct non-phosphorylated proteins and phosphorylated proteins than the traditional protein array technology and reverse protein array approach, only 80% to 90% of proteins detected by pathway array could be identified by traditional Western blot technique,②It is more sensitivity, the sensitivity can reach 1ng each belt when detected by chemiluminescence technology compared to traditional Western blot technique. It has repeatability. Therefore, this study used the following research pathway array technology.Objective:To explore the pathogenesis of ovarian cancer to seek for sensitive specific biological markers for early diagnosis, looking for molecular therapeutic target for ovarian cancer, provide experimental basis and theoretical basis for early diagnosis and treatment of EOC.Methods: (1)Research of differential proteomics of EOC were done through pathway array technology. Protein samples include ovarian cancer cell lines cultured in vitro and ultra-low temperature frozen tissue samples, then the proteins were separated by SDS-PAGE electrophoresis, transferred to NC membrane and then fixed the NC membrane with a Western blotting manifold that isolates 20 channels across the membrane, 2-4 kinds of antibodies which molecular weight differs from each other are blotted respectively in each channel, and western blotting were carried on as regular. Chemiluminescene exposure using Immun-StarTM HRP Peroxide Buffer/Immun-StarTM HRP Luminol Enhancer which is mixed together by the ratio of 1:1. Each membrane is evenly covered by 5 ml of the mixture. Capture imaging through one minute per exposure for 10 exposures after incubating with the mixture for 5 minutes. The NC membranes are analysed by Quantity One software Chemi XRS system to obtain images of different protein bands by chemiluminescence technology reflects the combination of the antibodies by specific proteins, and then by Chemi Doc system to make sure protein molecular weight corresponding to each phosphorylation proteins band or non-phosphorylated proteins band for protein characterization, and get relative expression volume of proteins by analysis of expression volume of each protein compared to the internal reference for quantitative comparison. In proteins from cultured cell lines, twice overexpression are defined significant difference while in proteins from tissues of EOC, SAM software are used to define significant difference. (2) Immunohistochemical staining and Western blotting technology were processed to clarify proteins over-expressed both in ovarian cancer tissue and ovarian cell line, which both further verified the results of pathway array.Results and discussion:(1)Differential expressed proteins in ovarian cancer cell lines (SKOV3 cells compared with IOSE 386 cells as an example):①6 over-expressed proteins: p-HER2/ErbB2 (Tyr1221/1222), p-PDK1 (Ser241), p-Akt (Ser473), TTF-1, ERK1/2 and p-Stat3 (Ser727) protein;②4 less-expressed proteins:p-RB (Ser780), p-p53 (Ser392), pc-Jun (Ser73), Cdc25B proteins. (2) Differential expressed proteins in ovarian cancer tissues compared with normal ovarian tissues:①12 over-expressed proteins: p-Stat3, p-PDK1, p-cdc2, p-p38, p-Akt, XIAP, PCNA, cdc2p34, p53, Cdk2, Mesothelin, E-cadherin proteins;②9 less-expressed proteins: ERK, Cdc25C, Wee 1, N-cadherin,α-tubulin, Vimentin, cdc42, Bax, PTEN protein levels are significantly decreased. (3)The study also uses immunohistochemical staining and Western blotting technology, further confirmed p-Stat3, Stat3, p-Akt, AKT, p-PDK1 over-expressed in ovarian cancer tissue compared with normal ovarian tissue, which both further verified the protein pathway array results. This shows that in vivo and in vitro ovarian cancer have started the same signaling pathway, the associated proteins should be the most prospects. However, differences proteins in ovarian cancer also showed that the expression of cell adhesion related proteins Mesothelin increased (8.29 fold), E-cadherin expression was increased (2..59 fold), X linked inhibitor of apoptosis protein XIAP expression increased 6.39 fold, the tumor suppressor gene protein P53 expression increased 21.50 fold. As can be seen from the above data the incidence of ovarian cancer in vivo complexity of the body by environmental constraints, so we will focus on the level of expression of ovarian cancer protein further studies to reveal whether there is a significant development value. With immunohistochemistry and Western Blot verification: both in SKOV3 ovarian cancer cell lines and in ovarian tissues, it was consistent with the high level expression of proteins which validate the results obtained with the pathway array. (4) With IPA system,we got top canonical pathways and networks for the 21 proteins.Conclusions: (1) Make up a new flat for screening of ovarian cancer by Pathway array technology, (2)12 kinds of differential expressed proteins in cell lines and 21 kinds of proteins in tissues were found by detected of 110 kinds of antibodies, (3) Proteins only expressed in tissue may have more significance for developing of ovarian cancer. (4)Preliminary revealed the signal transduction mechanism in the development of ovarian cancer by pathway array technology: PI3K/Akt pathway, JNK pathway, ERK pathway, p38 MAPK pathway, P53 pathways, cell cycle regulation disorders in the pathogenesis of ovarian cancer, shows the incidence of ovarian cancer involving multiple signal transduction pathways, (5) Akt, PDK1 could be markers for diagnosis.