Differential Expression Analysis Of Relation Of Ubiquitin-proteasome And Breast Cancer In Human

Objective Breast cancer is a much commonly diagnosed cancer and a leading cause of death for women. Although breast cancer has been studied extensively during the past several decades, the underlying molecular mechanism is currently not full understood. In both category and abundance, the expressed proteins in breast cancer cell are different from those in normal breast cell. To make the best of obtaining all information of proteins correlated to beast cancer, many medical workers used various kinds of samples to make breast cancer proteomic analysis in recent several years.We analyzed the differentially expressed genes and proteins between breast cancer tissue and adjacent normal tissue by the method of differential display to study the relation of breast cancer and ubiquitin-proteasome, which is favor of elucidating the underlying mechanism and searching for useful targets related to clinic for human breast cancerMaterials and methods The fresh breast cancer tissues and adjacent normal breast tissues were obtained at excision of surgical operation from 6 volunteers of 32-60 years old with breast cancer at different clinical stages [T1N1M0 (n=1), T1N2M0(n=1), T2N1M0 (n=4), T2N2M0 (n=3), T2N3M0 (n=1), T3N3M1(n=1), T3N2M0(n=1)] at the Affiliated Hospital of North Sichuan Medical College. All samples came from excised breast tissues after mastectomy. The breast cancer was pathologically identified as infiltrating ductal carcinoma. The adjacent normal breast tissues that were histomorphologically normal were selected, and fatty tissues were carefully removed.In this experiment, tissue protein extraction was accomplished by ReadyPre^TM Protein Extraction Kit (Total Protein) provided by Bio-Rad. two-dimensional polyacrylamide gel electrophoresis (2-DE) and mastrix-assisted laser desorption ionization time of flight tandem mass Spectrometry (MALDI-TOF-TOF MS) incorporated with online database research were performed to investigate differential proteins between breast cancer and adjacent normal tissues.Meantime, total RNA of samples from breast cancer and normal adjacent tissues were extracted respectively by using Total RNA Extraction Kit provided by WATSON BIOTECHNOLOGIES (Shanghai, China). Restriction fragment differential display polymerase chain reaction (RFDD-PCR) was performed to investigate up-regulation of expression of genes of USPs and PSs in breast cancer tissue compared adjacent normal tissue, and some of them were further verified as over-expression by immunohistochemical stain.Results Fourteen differential spots were identified with searching in an online database and MALDI-TOF-TOF MS analysis, and these differential spots had been detected in all samples from 12 volunteers with breast cancer. Of fourteen spots, 6 proteins were up-regulated (>4 folds), including elongation factor 1-beta (EF-1-beta), cathepsin D, translationally controlled tumor protein (TCTP), small ubiquitin-related modifier 3 precursor (SMT3A), ribosomal protein S12 (RPS12) and proteasome subunit alpha type 1 (PSMA1), 5 proteins were down-regulated, including serum albumin, immunoglobulin heavy chain gama, alpha-1-antitrypsin, apolipoproteinA-I (apoA-I) and immunoglobulin light chain, and 3 proteins (<4 folds) were possible to be up-regulated including superoxide dismutase (SOD), transthyretin (TBPA) and transgelin 2 in the infiltrating ductal carcinoma sample. Up-reguilation of genes of PSs and USPs (>3 folds) in breast cancer tissue compared to normal control were observed by restriction fragment differential display polymerase chain reaction (RFDD-PCR), including five PSs (PSMB5, PSMD1, PSMD2, PSMD8 and PSMD11), four ubiquitin specific proteases (USP9X, USP9Y, USP10 and USP25) and an ubiquitin protein ligase E3A (UBE3A). PSMD8, PSMD11 and UBE3A were further verified as over-expression by immunohistochemical stain.Conclusion By this experiment we verified that pathological changes of breast cancer are concerned with augmentation of substance metabolism, promotion of proteolytic activity, decline of activity of some inhibitors of enzymes and so on. Some important proteins which changes in expression are involved in pathological process of breast cancer may be useful biomarkers correlated to diagnosis, treatment effects and prognosis for patients with breast cancer, such as alpha-1-antitrypsin, EF-1-beta, Cathepsin D, TCTP, SMT3A, RPS12 and PSMA1. Of those, SMT3A, PSMA1 and RPS12 were firstly reported in this paper.Furthermore, by RFDD-PCR, MALDI-TOF-TOF-MS based on 2-DE and immunohistochemical stain, we verified that PSs and USPs are up-expressed in breast cancer tissue in both gene and protein levels, and the results suggested, in breast cancer, the activity of ubiquitin-proteasome system were obviously enhanced including ubiquitinating, degradation of proteins and deubiquitinating, and the underlining mechanism of breast cancer may be involved in the changes of functions of multifactors in multisteps related to ubiquitin-proteasome system.As observation and analysis of differentially expressed genes and proeins between breast cancer and adjacent normal tissue were done in this experiment, not only some import experimental data were obtained, but also some new ideas related to preclinical and clinical study of breast cancer may arise.