CRIP1生物学功能的定量蛋白质组学分析

摘要

CRIP1(cysteine-rich intestinal protein 1)是含有双锌指结构域的蛋白,在很多肿瘤细胞中高表达,但其在肺癌细胞中的生理学功能尚不明确.本研究应用定量蛋白质组学探究CRIP1过表达对肺癌顺铂耐药细胞(A549/DDP)的影响及作用机制.运用慢病毒载体系统构建了CRIP1过表达的A549/DDP稳转细胞系,利用Western Blotting证实了单克隆细胞系中CRIP1的过表达.发现CRIP1过表达能够加快细胞增殖,增加细胞的耐药性.通过定量蛋白质组学分析,鉴定了CRIP1过表达引起的蛋白质组的变化,并且对上调和下调的蛋白进行聚类分析.结果表明,CRIP1过表达上调了烟酰胺磷酸核糖转移酶(NAMPT)和NAD依赖型氧化还原酶的表达,从而促进细胞的增殖,并且提高了细胞的耐药性.%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.