Exploration Of The Function Of ZBTB9-a Member Of ZBTB Protein Family

The ZBTB family is a class of DNA binding proteins which contain the C₂H₂Küppel zinc finger motif and the BTB/POZ domain; they play many important roles invarious cellular activities, such as transcriptional regulatory, cell proliferation,differentiation, apoptosis, and functional regulation. We cloned a member of ZBTBfamily-ZBTB9（NM₁52735.3） by RT-PCR ananlysis depending on the sequenceinformation from database. Since its function remains unknown, so we carry outpreliminary exploration of the gene.The bioinformatics analysis results indicated that: ZBTB9was located on humanchromosome6p21.32; ZBTB9genomic structure is composed of two exons and oneintron, furthermore, and boundaries of the exon/intron were all consistent with theAG/GT rule; ZBTB9gene has a full open reading frame（ORF） of1421bp; ZBTB9geneencodes a protein with473amino acids; The molecular weight is predicated to be～50.6KD; SMART analysis showed that: ZBTB9protein contains a N-terminus BTB/POZ domain and two typical C₂H₂zinc finger motifs that extend to the end of theprotein sequence. Using biology software Clustal X, it might be concluded that humanZBTB9gene has closest affinity with that of Pan troglodytes，compared with that ofRat，Mouse，Macaca mulatta，Bos Taurus, Xenopus laevis based on the analysis ofmolecular evolution.When proteins are transported to specific subcellular compartments, they couldfunction. Subcellular fractionation showed that, ZBT9protein was localized in thenucleus, forming dot-like structures, ZBTB9protein lacking zinc finger motif waslocalized to around the cell nucleus, forming dot-like structures and ZBTB9proteinlacking BTB domain distributed mainly in the cell nucleus. The results suggest that theBTB domain plays a key role in spots like subcellular localization.In order to understand the transcriptional regulation fuction of ZBTB9protein, we performed Dual-luciferase reporter assay system to investigate the potential role ofZBTB9on signaling pathway. The results indicated that over expression of ZBTB9strongly enhanced the HSE luciferase activity. The further study on transcriptionalregulation function of different ZBTB9section demonstrated that, expression of eachtruncate could significantly enhance the HSE-luciferase activity, and transcriptionalactivation of ZBTB9protein lacking zinc finger motif was similar to that of ZBTB9protein; transcription activation of ZBTB9protein lacking BTB domain cansignificantely activate the HSE luciferase activity by nearly100%compared to that ofZBTB9protein. We speculate that ZBTB9may play transcriptional activation in thetranscriptional regulation of HSPs and HSE signaling pathway, thereby implicating in arange of life activities. Generally，the activation of HSE signaling pathway couldpromote the proliferation of malignant cells, and therefore ZBTB9is likely to functionin the proliferation of tumor cells.Studies have shown that the majority of ZBTB family members participate intumorigenesis and the development of the tumor. ZBTB9gene was obviously expressedin hepatoma cells compared to cervical cancer cells, glioblastoma cells, and gastriccancer cells. We will precisely analyze the effect of EGF and ATRA on ZBTB9’sexpression of human Hepatocellular carcinoma （HCC） cell line SMMC-7721byRT-PCR analysis. The results of RT-PCR showed that ZBTB9gene was expressed ineach group. However, there was high expression EGF group; ZBTB9gene were weaklyexpressed in ATRA group, thereby laying a valuable foundation for further study thefunction of ZBTB9in liver tumor.