Study On Molecular Regulatory Mechanisms Of Interferon β

Chronic hepatitis B (CHB) and chronic hepatitis C (CHC) are serious condition that often leads to complications of progressive liver diseases including cirrhosis and hepatocellular carcinoma. Treatment with alpha-interferon (IFN α ) has shown to arrest the progression of this hepatic injury. However, IFN α is only effective in 30%~40% of patients. Lamivudine, adefovir and ribavirin have been approved for use in HBV and HCV infection and evaluated in long term clinical trials. Although lamivudine, adevofir therapy have favorable effects on virologic, histologic and biochemical features of disease, a high rate of viral resistance limits the efficacy of these treatments. Therefore, further attempts need to be done for non-responder patients to alpha-interferon or failed to respond to alpha-interferon therapy. Recently, some clinical trials have shown that other alternative might be the administration of beta-interferon (IFN β ).Production of IFN β is mainly induced in fibroblasts by viral and other foreign nucleic acids. IFN β is an important cytokine with diverse biological function, the peculiar efficacy of IFN β has been demonstrated in the treatment of patients with chronic hepatitis B and C, who did not respond to IFN α . Moreover, antitumor effect of IFN β when combined with anticancer drugs are more greater than those of IFN α in treatment of HCC. However, the exact molecular mechanisms for IFN β transcriptional regulation, antiviral and binding protein in the hepatocyte are still not well known.Studies on the regulation of gene expression by IFN β will help us understand the mechanisms of IFN β. To explore the trans-regulation effects of IFN β, the recombined expression plasmid, pcDNA 3.1(-)-IFN β was constructed and transfected into HepG2 cells. pcDNA 3.1(-) empty vector was used as control. The mRNA was isolated from HepG2 cells transfected with pcDNA 3.1(-)-IFN β and pcDNA 3.1(-) empty vector, respectively, and suppression subtractivehybridization (SSH) method was employed to analyzed the differentially expressed DNA sequence between the two groups. The obtained product was subcloned into T/A plasmid vectors to set up the subtractive library. Amplification of the library was carried out with E. coli strain DH5 a . The DNA was sequenced and analyzed in GenBank with Blast search after PCR. At the same time, SSH method and bioinformatics techniques were also used for screening and cloning of the differentially expressed target genes in HepG2 cells induced by recombinant IFN P and 0.9 percent sodium chloride. SSH clony PCR showed that altogether 25 up-regulation coding gene sequences were obtained, including two unknown function gene;23 down- regulation coding gene sequences, including a unknown function gene.Gene chip technique was employed for detecting and analyzing of mRNA from the HepG2 cells transfected and recombinant IFN P induced. The obtained sequences may be target genes up and down-regulated by IFN P , and some genes coding proteins involved in cell cycle regulation, signal transduction, translation and synthesis of protein, correlated with tumor and metabolism.IFN P differential gene expression is modulated by a complex interplay between cis-acting DNA elements and corresponding specific trans-regulating factors. In this study, we screened proteins that could bind with IFN P promoter from liver cDNA library by phage display technique. IFN P promoter was coated on the microplate and incubated with cDNA library for phage display. Phages bound with IFN P promoter were enriched and positive clones were amplified after five rounds biopanning. DNA sequence analysis and subsequently compouter blasting analysis showed that proteins capable of binding with IFN P promoter were as follows: glutathione S-transferase omega 1 (GSTO1), schistosoma mansoni ubiquitin mRNA, Brugia malayi IL-5 receptor binding protein mRNA,synthetic construct mutant a -amylase, syntheic construct zinc finger protein 42 (myeloid-specific retinoic acid-responsive) mRNA, fetal Alzheimer antigen (FALZ), transcript variant 1, and PHD finger transcripton factor mRNA.Yeast-two hybrid technique was used to screen binding proteins of interferon P (IFN P ) from human hepatic cDNA libraty. The IFN ￡ gene was amplified by polymerase chain reaction (PCR) and IFN 3 plasmid was constructed with yeast-two hybrid system. pGBKT7-IFN P was transfected into yeast AH 109. The transfected yeast was mated with yeast Y187 containing liver cDNA library plasmid in 2*YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X- a -gal for selecting. After plasmid extracting and enzyme digest analysis, the blue colonies were performed sequence analysis, the results were analyzed by bioinformatics. IFN $ gene was successful cloned and expressed in yeast cells. Nineteen genes in twenty-nine positive colonies were obtained, coding 15 kinds of proteins, including 1 unknown function protein.In some extent, these proteins with different functions may provide some possible clues for mechanism of IFN & in vivo.