Quantitative Proteomics Analysis on Biological Function of CRIP1

The aim of the present study is to investigate the effects of cysteine-rich intestinal protein (CRIP1) over-expression on cellular processes using proteomics. CRIP1 is a protein that contains a double zinc finger domain, and is highly expressed in many tumor cells, but its roles in lung cancer cells remain elusive. In this study, the CRIP1 gene was cloned and used pLVX-IRES-Zsgreen1 lentiviral vector that encodes a GFP to transfect A549/DDP cells that was a cisplatin resistant cells as compared to A549 cells. The real-time quantitative PCR (qPCR) was used to confirm the transfection efficiency of CRIP1. Flow cytometry was used to isolate single cells, which was seeded into a 96-well plate and cultured to obtain monoclonal cell lines. The monoclonal cell lines with high brightness were screened by fluorescence microscopy and CRIP1 over-expression was confirmed by western blot analysis, indicating that a stable cell line was established successfully, in which CRIP1 was over-expressed. The growth curves of CRIP1 over-expression cells and the control cells were measured by the CCK8 assay. It showed that CRIP1 over-expression increased cell proliferation. CRIP1 over-expression cells and the control cells were also treated with cisplatin at different concentrations for 24 h and examined the viability of cisplatin-treated cells. Results showed that CRIP1 over-expression enhanced cells’ resistance to cisplatin.
To further explore the mechanism underlying CRIP1 over-expression mediated cellular processes, quantitative proteomics was applied to identify differentially expressed proteins between the control and CRIP1 over-expression cells. The quantitative proteomics was carried out using the TMTsixplex^TM Isobaric Label Reagent, which modified the free amino group at the peptide N-terminus and lysine residues. Modified peptides generated reporter ions in MS/MS spectra to provide quantitative information about the proteins being analyzed. It was found that CRIP1 over-expression upregulated the expressions of nicotinamide phosphoribosyltransferase (NAMPT), which was the rate limiting enzyme in the scavenge pathway for production of the intracellular nicotinamide adenine dinucleotide (NAD). The CRIP1 over-expression mediated upregulation of NAMPT can increase the cellular NAD level. It was also revealed that CRIP1 over-expression upregulated expressions of NAD-dependent oxidoreductases, such as aldo-keto reductase family 1 member C1 (AKR1C1) and aldo-keto reductase family 1 member B10 (AKR1B10). Extensive studies have reported that NAD is essential for the cellular anti-oxidant systems and for maintaining mitochondrial integrity. The results suggest that the CRIP1 over-expression mediated NAMPT upregulation contributed to the enhanced cell growth and resistance to cisplatin.