Serial Analysis Of Gene Expression In Human Hepatoma Cell Line HepG2 Induced By Paeonol-Arsenic Compound

Hepatocellular carcinoma(HCC) is one of the most common malignant tumors, and has become the second leading killer in cancer. There is a lack of efficient drugs for HCC therapy, so discovery and screening new drugs for the treatment of liver cancer is essential and important. In now days with the rapid development of molecular oncology, the screening of anticancer drugs is converted from that aiming at diseases to that aiming at mechanism. It is key in search of specific molecular targets for therapy, i.e. discovery of the biochemical and molecular difference between cancer and normal cells. Therefore, if in deep research of the mechanism of hepatocarcinogenesis, it will try to find out the method of analysis and estimate of action mechanism of anti-cancer drugs, providing a new strategy for the screening of anti-cancer drugs.In this study, the genes differentially expressed in human hepatoma cell line HepG2 induced by paeonol-arsenic compound, 2-methoxy-4-hydroxy-5-acetyl-azobenzene -4-arsenic acid (R2), were analyzed qualitatively and quantitatively with long serial analysis of gene expression (LongSAGE) protocol, and then the genes expression-profile was.established. We try to find out the molecular targets, and discuss the molecular mechanism of paeonol-arsenic compound acting on hepatocellular carcinoma.Total RNA was isolated from HepG2 induced by paeonol-arsenic compound. The cDNA was synthesized from mRNA immobilized on magnetic beads. The cDNA was then cleaved with a restriction enzyme called NlaIII, and the cleavage product was divided equally into two portions. Adapter A and Adapter B were ligated to each portion using Nlalll cohesive termini, then the tags were released with digestion of tagging enzyme Mme I. The tags linked together to establish the 130bp ditags by T4 DNA ligase. The products were scale-up PCR-amplified to obtain adequate 130bp ditags, and were cleaved with Nlalll again to produce 34bp ditags. The 34bp ditags were ligated to obtain concatemers. The concatemers were cloned into pZErO(?)-1 vector, transformed and ready to sequence. The raw sequencing files were analyzed by SAGE2000 software, and a LongSAGE library was constructed from HepG2 induced by paeonol-arsenic compound. The LongSAGE library was compared with hepatocellular carcinoma (HCC) library from the SAGEmap, in order to find out the differentially expressed genes.A total of 675 tags were extracted from the 45 the sequenced files, representing401 unique tags. Of these tags, 263 (66%) tags occurred once and 57 (14%) tags were identified multiple times. 81 (20%) tags were not reported before. Between the LongSAGE library and HCC library, there were much difference of gene expression, such as PAFAH2, MCM3, CTPS, GFM2, HSP70, TMSB4X , TXNRD1, CYP3A4, MAP2K2, SET, CASP8, OXCT1 et al. The results indicate that R2 can down-regulate the expression of PAFAH2,MCM3,CTPS,GFM2,HSP70, up-regulate the expression of TMSB4X, TXNRD1, CYP3A4, MAP2K2, SET, CASP8, OXCT1. It is also suggested that R2 may play important roles in the anti-tumor effect.SAGE can provide the full and quantitative information of gene expression, identify the molecular targets of paeonol-arsenic compound, and help to clarify the molecular mechanism of paeonol-arsenic compound. It further indicates that paeonol-arsenic compound may be a potential anti-tumor drug.