Summary

Title

PGRMC1 is a novel potential tumor biomarker of human renal cell carcinoma based on quantitative proteomics and integrative biological assessments

Description

Progesterone receptor membrane component 1 (PGRMC1) is widely observed at elevated expression levels in multiple human cancers. However, the associations of PGRMC1 with renal cancer are not clear and merit further study. In this report, we report a systematic, integrative biological assessment of PGRMC1 in renal cell carcinoma (RCC), encompassing quantitative proteomics, immunohistochemical profiling, and its clinicopathologic significance. We identified that PGRMC1 is increased to 3.91-fold in RCC tissues when compared with its autologous para-cancerous tissues by a quantitative proteomic analysis. PGRMC1 was widely increased in 63.7% RCC samples (86/135) by immunohistochemical validation. Meanwhile the average expression level of serum PGRMC1 in RCC patients (n=18) was significantly increased to 1.67-fold compared with the healthy persons. Moreover PGRMC1 upregulation is correlated with tumor malignancy level and a poor overall survival for RCC. And PGRMC1 overexpression promotes cell proliferation for renal cancer cells in vitro. Our findings demonstrate that PGRMC1 is a novel potential biomarker and therapeutic target for renal cancer due to PGRMC1 roles in promoting RCC-associated phenotypes in vitro and in vivo.

Sample Processing Protocol

HEK293 cells were cultured in deuterated-leucine (Leu-d3) (5, 5, 5-D3, 98%; Cambridge Isotope Laboratories, UK) containing DMEM medium with 10% dialyzed fetal bovine serum (FBS) (GIBCO). Tissue protein was respectively extracted from renal cancer tissue (RCT) and its para-cancerous counterpart (PKT) according to our previous approach . The RCT proteins were extracted and evenly mixed from 3 RCTs to avoid individual sample variance. Similarly 3 PKTs were blended together to extract tissue proteins as follows. Tissues were cut into small pieces, mixed with liquid nitrogen to grind into powder. Then RIPA buffer was added to extract tissue proteins by 15 000-rpm centrifugation for 30 min at 4℃. The labeled HEK293T cells were collected to dissolve with RIPA buffer to extract cellular proteins by centrifugation. Protein concentration extracted from HEK293 cells, RCTs and PKTs was respectively quantified using the Protein Assay Kit (#500-0006, Bio-Rad, Hercules, CA, USA). Protein concentrations were usually in the range of 6–12 mg/ml.The pooled PKT or RCT proteins were respectively mixed with the Leu-d3-labeled HEK293 cellular proteins at 1:1 ratio, which was named protein sample 1 and protein sample 2 in the following SILAC-MS/MS analysis. The protein sample 1 was composed of Leu-d3-labeled proteins from HEK293 and unlabeled proteins of PKTs, and the protein sample 2 was derived from Leu-d3-labeled cellular proteins combined with unlabeled proteins of RCTs. Then the protein samples were separated on a 12% SDS-PAGE and Coomassie stained to visualize gel bands. The gels were excised and subjected to in-gel digestion overnight using mass spectrometry -grade trypsin (Promega, Madison, WI, U.S.A.) . The peptides were extracted and prepared for MS/MS identification,Two independent MS/MS identification experiments were performed for each protein sample.

Data Processing Protocol

Mascot sever was used as the database search engine for peptide identifications against the uniport human database. Protein isoforms and proteins that cannot be distinguished based on the peptides identified are grouped. The identification of proteins was set to a false discovery rate of 1%. Only proteins that were identified in both biological samples were included in further analysis. The parameters for database searching were set as following: (i) the mass tolerance was set as 0.3 Da for MS and MS/MS; (ii) trypsin enzyme specificity and two max-missed cleavages were allowed; (iii) variable modifications included oxidation of methionine and Leu-d3 labeling.The SILAC ratio of a protein in the protein sample was defined as the isotope peak intensity ratio of the unlabeled peptides from tissue proteins versus the Leu-d3-labeled cellular peptides from HEK293 cells. At least one Leu-containing peptide was used to quantify protein expression in SILAC-MS analysis, and the protein expression

Contact

Xixi Wang, Sichuan University
Shufang Liang, State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy ( lab head )

Submission Date

19/07/2016

Publication Date

24/01/2017

Cell Type

kidney cell

Instrument

QSTAR

Software

Not available

Quantification

SILAC

Publication

    Zhang D, Xia X, Wang X, Zhang P, Lu W, Yu Y, Deng S, Yang H, Zhu H, Xu N, Liang S. PGRMC1 Is a Novel Potential Tumor Biomarker of Human Renal Cell Carcinoma Based on Quantitative Proteomic and Integrative Biological Assessments. PLoS One. 2017 Jan 20;12(1):e0170453 PubMed: 28107520