Initial Exploration Of The Function Of ZBTB8A-a Member Of ZBTB Protein Family

The ZBTB proteins which are important member of the zinc finger protein family that function as transcription factors play important roles in various cellular functions, such as transcriptional regulation, cell proliferation, differentiation and apoptosis. Firstly, we cloned a gene , named as ZBTB8A which is a member of human ZBTB family, from gastric cancer cell AGS cDNA by RT-PCR. Since there are no reports on ZBTB8A, we carry out preliminary exploration on this gene.Bioinformatics databases were used to analyze and predict its structure and function. Results showed that ZBTB8A(NM₀01040441) gene has a full open reading frame (ORF) of 1326bp, which was localized on chromosome 1p35.1. The ZBTB8A gene encodes a protein with 441 amino acids. The molecular weight is predicted to be 50 kDa. SMART analysis showed that ZBTB8A protein contains a N-terminal BTB/POZ domain and two typical C2H2 zinc-finger motifs that extend to the end of the protein sequence. Comparing BTB/POZ domains derived from ZBTB8A with other known proteins, we found that the BTB/POZ domain of ZBTB8A is highly homolous to the BTB/POZ domain of other known proteins.To verify the subcelluar location of ZBTB8A protein, we contructed the plasmid pEGFP-ZBTB8A which express the fused protein EGFP-ZBTB8A. The results indicated that ZBTB8A was localized in the nucleus, forming dot-like structures. Further study showed that the BTB domain was required for spots like localization, and the zinc finger domain was required for nuclear localization of the ZBTB8A protein.Many studies showed that many ZBTB proteins functioned as transcription factors. We applied pGal45tkLuc vector to examine the potential function of ZBTB8A in transcriptional regulation.We found that ZBTB8A might play a role of transcriptional repressor in mammalian cells. Furthermore, we performed Dual-luciferase reporter assay system to investigate the potential role of ZBTB8A on signaling pathway including AP1, NFAT, MYC, Rb, HSE, CRE, AP1(PMA), GRE and NF-κB. It was indicated that ZBTB8A functioned as a dose-dependent suppressor of the CRE reporter gene.The further study on function of differert ZBTB8A section showed that the BTB domain and zinc finger domain in the inhibition of CRE transcription was very important because lacking of either domain will significantly reduce the inhibition effect. The pCRE-luc plasmid which contains CRE element is designed to monitor the CRE-mediated signal transduction pathways. ZBTB8A may have a series of inhibition in CRE-mediated signaling pathway to regulate cell function.Four ZBTB8A interference vectors were designed and synthesized for further study. Moreover, we identified effective ZBTB8A interference expression vector by semi- quantitative PCR. The stably cell lines transfected by sense vectors or interference vectors were sieved by G418. All of the related work will be helpful for further research of ZBTB8A in AGS gastric cancer cells, and determining ZBTB8A function in tumor cells.