Differential Protein Expression In Serum Of Cervical Carcinoma And Preliminary Proteomics Study Of HeLa Cells After Radiation Therapy

Objective:1.To explore the serum protein mass spectrum of the cervical intraepithelial neoplasia (CIN) III, cervical carcinoma and healthy women to screen its potential special biomarker by surface-enhanced laser desorption/inionation-time of flight-mass spectrometry (SELDI-TOF-MS) and weak cation exchange (WCX) chip, and to evaluate its clinical significance.2. To screen and identificate differential expression proteins of Hela cells after radiation therapy, and to analyze the molecular mechanism of Hela cell after radiation therapy at the level of proteomics.Methods:1. Identify the differentially expressed proteins in the serum of CINⅢ, cervical carcinoma patients in earlier stage and healthy women(1) Sample collection:23 CINⅢpatients,34 cervical carcinoma patients in earlier stage and 35 healthy women as control. Collect the venous blood of all women. Centrifuge blood at 4℃and take the supernatant which was preserved infrigidaire at-80℃.(2) Serum protein mass peaks were detected by SELDI-TOF-MS and weak cation exchange chips. Fetch array data and draw protein mass spectra figure. The protein fingerprints were obtained, Biomark Wizard software was used to detect the differently expressed proteins, and the differently expressed proteins were searched in the Swiss protein database.(3) Receive Operating Characteristic (ROC) curve was employed to evaluate the clinical value of the differently expressed proteins. 2. Proteomics analysis of HeLa cells after radiation therapyHela cells were exposured to 6Gy doses of 6MV X-ray at one time. The total proteins of Hela cells with pre- and after-radiation therapy were obtained, and were extracted and separated by two-dimensional gel electrophoresis(2-DE). The images of the gels were aequired by the scanner and then analyzed by using PDQuest 8.0.1 software to find the differentially expression protein-spots in each group. Then the differentially expressed protein-spots were incised from the gels and digested by trypsin. The peptide mass fingerprintings(PMF) were acquired by matrix assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) and the proteins were identified by data searching in the Mascot-database.Results:1. Seventy-six differential protein peaks were detected in all the serum. In the range of mass-to-charge ratio (m/z) 2000-20000Da in the groups of cervical carcinoma and healthy women, ten statistical differently expressed proteins were identified including five up-regulated proteins with m/z of 2951.5,3882.4,5310.3,7757.1 and 15910.4 respectively in the cervical carcinoma patients in earlier stage, five down-regulated ones with m/z of 3824.6,4211.8,4512.4,4786.4 and 5565.3 respectively. The sensitivity and specificity of diagnosis of the cervical carcinoma of calculated each differently expressed protein were 64.7%～97.1%and 79.4%～94.3%, respectively. There were three up-regulated proteins in the serum of CINⅢcompared with the cervical carcinoma group with m/z of 4584.9,5897.5 and 9176.6.2. The proteins were separated sueeessfully for proteome research,2-DE maps of total proteins with high resolution and reproducibility from Hela cells were established. The average spots of Hela cells after-radiation and the pre-radiation group were (1248±67) and (1207±78) respectively, and 86.8% spots between the two groups were matched. Ten differentially expressed protein spots were chosen and identified by MALDI-TOF-MS. There were six up-regulated proteins in the Hela cells after-radiation group:Keratin 10, Keratin 18, Annexin, acidic ribosomal protein, Lupus La protein and Arginine deiminase; four up-regulated proteins in the Hela cells pre-radiation group:Calreticulin, Phosphoglycerate mutase 1 and Succinate dehydrogenase flavoprotein.Conclusions:1. Differential protein expression exists in healthy women, CINⅢand cervical carcinoma patients. The potential protein biomarkers could be obtained by SELDI-TOF-MS coupled with WCX chips with good sensitivity and specificity in the serum of cervical carcinoma patients and healthy women, which is helpful tool for the screening and earlier diagnosis of cervical carcinoma.2. Radiationtherapy may change the cell proliferation and differentiation through affecting protein expression which affected the apoptosis, cell cycle, and signal transduction. Further study on the signal transductions and their relationship may not only be helpful to explore the molecular mechanism of Hela cells after radiation therapy at proteomics level, but also provide a new idea for the molecular treatment of cervical carcinoma.