SILAC-based Quantitative Proteomic Analysis Of KLK7-induced Prostate Cancer Cells

OBJECTIVE To construct the KLK7 overexpressed prostate cancer cell line 22RV1-KLK7, detect the differentially expressed proteins betwee 22RV1-KLK7 cells and control cells using the MS-based method of stable isotope labeling with amino acids in cell culture (SILAC) approach, analyze the KLK7-regulated protein expression profiles, and conduct experimental verification of the candidate proteins; To provide the molecular basis of protein to investigate the biological function and mechanism of KLK7 in prostate cancer progression.METHODS 1. We constructed KLK7-overexpressed cell line 22RV1- KLK7 using plasmid transfection approach, mRNA and protein levels were experimental verified; 2. 22RV1-KLK7 cells were cultured in the heavy isotope labeling amino acid medium, 22RV1-V cells were cultured in normal medium.Protein of two groups were extracted respectively, after quantitative determination, SDS-PAGE electrophoresis and Coomassie blue staining,dehydration, hydrolysis, peptide extraction, and lyophilized, the liquid chromatography-mass spectrometry (HPLC-MS/MS) for protein, two sets of samples were identified and quantified; 3. Getting two mass search library results of variance analysis, and obtaining 22RV1-KLK7 differential protein profiling; 4. Using bioinformatics analysis to find the main functions classification, signaling pathways, and interaction networks of differential expressed proteins, providing a theoretical basis for KLK7 biological functions in PCa progression ; 5. Selecting significant candidate proteins to perform verification experiment at mRNA and protein levels; 6. Selecting significant candidate proteins and validating in tissue microarray chips by immunohistochemistry (IHC) at protein level.Meanwhile, the correlations between the expression of these proteins and clinicopathological features and PCa prognosis will be analyzed to look for clinical indicators related to these proteins.RESULTS 1. 22RV1-KLK7 cell lines which stably overexpress KLK7 protein was successfully constructed, providing experimental material for subsequent proteomic experiments; 2. In all, 2006 proteins were identified and quantified,which gave us 350 differentially expressed proteins (P<0.01, fold change ratio >1.3 or <0.77); among them, 137 proteins were up-regulated and 213 were down-regulated. 3. Bioinformatics analysis showed that functional classification of identified up-regulated proteins mainly focus on response to chemical stimulation, stress response, negative regulation of apoptosis, actin filament-based processes, down-regulated proteins focus on metabolism,biosynthesis, and oxidation reduction; 4. We choose 2 candidate proteins (AGR2 and TST) to validate the expression at mRNA level, the result of RT-q PCR showed that the AGR2 and TST mRNA level of 22RV1-KLK7 were significantly increased (21.8±6.31 VS 1.21 ± 0.21,P<0.01; 36.7±7.23 VS1.09 ± 0.16, P<0.01); Results of western blotting showed that the expression of AGR2 and TST proteins were significantly increased in 22RV1-KLK7; 4. The result of IHC showed that AGR2 and TST proteins are highly expressed inadenocarcinoma tissues, but not in BPH or normal prostate tissue, suggesting that AGR2 and TST could be used as candidate molecular markers for PCa.CONCLUSIONS 1. Our study successfully constructed overexpressed KLK7 prostate cancer cell line 22RV1-KLK7; 2. A network of KLK7 regulated protein profile of 22RV1 cells was established for the first time, and the biological functions of these proteins were preliminary explored. We consider that KLK7 could promote the invasion and metastasis of tumor cells; 3. We verified the mRNA and protein level of AGR2 and TST in 22RV1-KLK7 cell line,confirmed the reliability of the results of the proteome; 4. We further explored the expression levels of AGR2 and TST in section specimens of clinical samples,AGR2 and TST were up-regulated in adenocarcinoma and high expression of AGR2 closely correlated with lower tumor stage.