关键词: 关键词：PCBP2, 微RNA, RIP-Chip, 神经胶质瘤

Abstract: Objective To screen all the microRNAs (miRNAs) bound to poly(C) binding protein 2 (PCBP2) in normal human astrocytes (HA) and glioma cells (T98G and U87MG) using ribonucleoprotein immunoprecipitation - microarray profiling (RIP-Chip). Methods The expression levels of PCBP2 protein were detected in HA, T98G and U87MG cells by Western blot analysis with the PCBP2 antibody purified from rabbit serum. With normal rabbit IgG as negative controls, the protein and RNA samples from RIP assay were enriched in the three cell lines. The protein samples were detected enrichment effect by Western blot; The puried RNAs were quantified by the NanoDrop ND-2100 and the RNA integrity was assessed using Agilent 2100.The labeled RNAs were hybridized onto the Affymetrix miRNA 3.0 microarray. The enriched miRNAs were then identified through fold change as well as P value calculated using t-test. The threshold set for up - regulated genes was a fold change > 4 and a P value < 0.05. Results RIP-certified anti-PCBP2 antibody was validated for use in ribonucleoprotein (RNP) immunoprecipitation (RIP) in conjunction with the RIP-Assay Kit for microRNA. Its ability to immunoprecipitate RNP complex and miRNAs was confirmed. 103 mature miRNAs, 1 precursor miRNA (pre-miRNA) and 1 small nucleolar RNA (snoRNA) were selected to interact with PCBP2; 15 of which exist the PCBP2 binding sites. Conclusions PCBP2 can bind to mature miRNA, pre-miRNA or snoRNA in vivo not only through the target sequence recognition but also by the formation of ribonucleoprotein complexes.

Key words: Key words: PCBP2, microRNA, RIP-Chip, glioma