ZNF191 Targets Upregulation Of Beta-catenin To Promote Hepatocarcinoma Cell Proliferation

ZNF191 is a new human C2H2 type zinc finger protein gene first reported by our lab in 1997. Tissue mRNA analysis showed that ZNF191 gene was ubiquitously expressed in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, pancreas, etc. Our previous research revealed that ZNF191 was notebaly up-regulated in HCC, and in this study we confirmed the result by using real-time PCR and western blot analyses. In order to explore the role of ZNF191 in HCC, we searched for ZNF191 target genes by using a transient knockdown strategy in human HCC cell line L02 with microarray (Affymetrix HG-133 Plus2.0) analyses, and confirmed the result by real-time PCR. Several candidate genes such asβ-Catenin were further researched to elucidate transcription mechanism of downstream genes of ZNF191 and biological implication of them in HCC.With detailed analysis of gene expression pattern in transient ZNF191 knockdown cell line L02, we identified that significantly altered genes are involved in processes of the regulation of cell cycle, signal transduction, transcription, zinc ion binding, cartilage/skeletal development, metabolic process, etc. The findings reveal that ZNF191 is a pleiotropic factor in cellular process.To our great interesting, several of the altered genes are involved in Wnt signal pathway, a very important pathway in cell. Among themβ-Catenin mRNA was down regulated to 0.33 fold and that of CyclinDl, the downstream gene ofβ-Catenin, was down regulated to 0.5 fold.β-Catenin and CyclinDl proteins were also down regulated in transient ZNF191 knockdown L02 cell with western blot analysis. With transient over expression of ZNF191 protein,β-Catenin and CyclinDl proteins were up regulated in L02 cell. Then we constructed stable ZNF191 knockdown cell lines L02 and Hep3B, and found thatβ-Catenin and CyclinDl proteins were down regulated, the number of cells in the S phase decreased, and cell proliferation was significantly inhibited. Thus ZNF191can positively regulateβ-Catenin, and consequently influence cell cycle and cell growth.Promoter luciferase assay indicated ZNF191 can increase transcription activity of full-length CTNNB1 promoter, as well as that of CCND1 promoter in a dose-dependent manner. While mutant in LEF/TCF site of CCND1 promoter resulted in a much lower increase in transcription activity. With delicate analysis of various lengths of CTNNB1 5’-flanking region, it shows that the promoter region of CTNNB1 is located at nt-1407/-907 which has the maximum transcriptional activity. EMSA assay indicates purified ZNF191 protein can directly bind to CTNNB1 promoter, and the binding region of CTNNB1 promoter is located at nt-1254/-1224, and the key sequence of binding site is ATTAATT.In summery, our work demonstrates ZNF191 is up-regulated in HCC. We discovered firstly that ZNF191 can promote Wnt signal pathway with microarray analyses of gene expression pattern in transient ZNF191 knockdown cell line L02. ZNF191 can directly bind to CTNNB1 promoter, increase transcription ofβ-Catenin and CyclinDl, and promote cell proliferation of HCC. Thus ZNF191, an up-regulating gene in HCC, is a potential target for diagnosis, therapy and new drug development of HCC.