Role Of ZNF403in Cancer

ZNF403, also known as GGNBP2(gametogenetin bindingprotein2) is mapped to chromosome location,17q12-21.1, a region associated with cancers such as breast cancer, prostate cancer, and laryngeal cancer ZNF403is highly conserved from drosophila to human. The mouse and human ZNF403share96%amino acid sequence identity. In humans, there are two known RNA transcripts produced from ZNF403. The short transcript, laryngeal carcinoma-related gene1(LCRG1) was originally identified in human laryngeal carcinoma by mRNA differential display LCRG1encodes a nuclear protein of288amino acids, corresponding to a C-terminal truncation of ZNF403. It has been reported that over-expression of exogenous LCRG1suppresses the growth rate of human laryngeal cell lines.The full size ZNF403transcript is translated into a protein of696amino acids, which is the focus of the current study. However, the exact biological function of ZNF403is not clear.To investigate the role of ZNF403, we used the helper dependent adenoviral vector (HD-Ad) mediated RNA interference technology and conducted loss-of-function analysis of ZNF403in human tumor cells. U6promoter based vector was utilized to express ZNF403-targeted shRNA, which was then inserted it into HD-Ad vector. With the existence of helper virus, we successfully produced HD-Ad-ZNF403-shRNA of high purity and high efficiency by Cre-loxp system after rescue, amplification, large scale production and purification by CsCl gradient super centrifugation. Furthermore, we used HD-Ad-ZNF403-shRNA to knockdown expression of endogenous ZNF403and next characterized the impact of ZNF403knockdown on cell proliferation on cell proliferation, anchorage-independent growth and cell migration in human tumor cells. Moreover, we conducted tumor growth in nude mice and validated its role on cell proliferation in vivo. To elucidate the mechanism of ZNF403in cell proliferation, flow cytometric analyses of cellular DNA contents using Propidium Iodide and BrdU, indictive of DNA replication during S phase, were carried out to examine the role of ZNF403on cell progression. Subsequently, human cell cycle realtime PCR array with the advantages of high-throughput and accuracy were applied to further decipher the mechanism underlying the G2/M cell cycle arrest by ZNF403knockdown. We compared expression level of84cell-cycle genes between ZNF403knockdown group and control group in Hep-2and HEK293cells and validated several important altered genes by western blot analysis, which shed new light on the role of ZNF403in the regulation of cell cycle progression.Since it has been reported that ZNF403can be induced by TCDD through AHR pathway, in order to reveal the molecular mechanism underlying ZNF403’s induction by TCDD, we made use of bioinformatics analysis and investigated the transcription regulation of ZNF403. We first analyzed the5’upstream of ZNF403gene using bioinformatics and predicted the location of promoters and transcription binding sites. A series of promoter deletions within luciferase reporter genes were then constructed and dual-luciferase assay were performed in various cell lines to identify, localize and characterize the ZNF403promoter. Importantly, we accessed the transcription regulation of TCDD on its promoter. These findings suggest a previous unrecognized role of ZNF403in cell proliferation, which led to better understanding of the molecular mechanism on the role of ZNF403in tumorigenesis.[Gene structure,evolution and endogenous expression analysis of human ZNF403gene]ZNF403is mapped to chromosome location,17q12-21.1, a region associated with cancers such as breast cancer, prostate cancer, and laryngeal cancer. In human, there are two known RNA transcripts produced from ZNF403. The full size ZNF403transcript is translated into a protein of696amino acids, which is the focus of the current study. The length of the full size transcript contains2689bps, including14exons. The coding region spans from317-2410. The size of the short transcript, Laryngeal carcinoma related gene1(LCRG1) is3448bps including7exons and encodes a nuclear protein of288amino acids. Evolutionary comparisons of13vertebrate genomes by ECR browser, the human ZNF403gene shares highly phylogenetic homology to some extent with chimpanzee, rhesus monkey, dog, cow and mouse ZNF403genes, suggesting it is a highly conserved gene during evolution. To understand the relative expression level of ZNF403and LCRG1, we performed real-time PCR in different cell lines. The expression level of ZNF403is significantly higher (>10folds) than that of LCRG1in Hep-2, HeLa, Jurket, BEAS-2B, HEK293cell lines. At protein level, only ZNF403can be detected by western blot. Although the level of ZNF403varied in these cell lines, the expression pattern of ZNF403and LCRG1was similar. These data suggest that ZNF403is the major transcript produced from ZNF403gene.[Design, construction, identification and efficiency test of helper dependent adenoviral vector mediated ZNF403-targeted RNA interference system]RNA polymerase Ⅲ, U6promoter-based DNA vector was used to express shRNA. A ZNF403target region was chosen beginning with three guanines (51-GGGCAAATTCTGAAGAGAACGACA-3’). Two pairs of complementary DNA oligos were used to make the shRNA construct. The U6promoter and the ZNF403shRNA sequence was subcloned into the HD-Ad vector pC4HSU. The viral vector is linearized by Pme I to expose the viral ITRs and used to transfect116cells for virus rescue and amplification. The HDAd vectors were produced by Cre/loxP system. To rescue the HDAd, the linearized genome is transfected into116cells expressing Cre and infected with a helper virus (HV) providing a packaging signal flanked by loxP sites. After rescue, amplification and large scale production, producer cells containing a large amount of HD-Ads were harvested and digested by RNase A and DNase I. Finally, CsCl gradient super centrifugation and dialysis were performed to remove the helper virus and purify HD-Ad vectors. Southern blot and OD measurement showed that we achieved the production of highpurity HD-Ad-ZNF403-shRNA amounted to10^13.To test the silencing efficiency of the HD-Ad-ZNF403-shRNA, we transduced Hep-2and HEK293cell lines with HD-Ad-ZNF403-shRNA and HD-Ad-shRNA-control at various concentrations, real-time PCR analysis and western blot analysis showed that the expression of ZNF403was decreased dramatically at50MOI after HD-Ad-ZNF403-shRNA transduction. No difference was found in the expression of LCRG1between the cells treated with HD-Ad-ZNF403-shRNA and those with shRNA-control. Thus, HD-Ad delivery of shRNA effectively and specifically knocks down endogenous ZNF403and provides a powerful tool for loss-of-function analysis of ZNF403without interfering with expression of LCRG1.[Characterization impact of ZNF403knockdown on human laryngeal carcinoma Hep-2cells] HD-Ad-ZNF403-shRNA and none-effect HD-Ad-shRNA-control were used to transduce Hep-2and HEK293cells at50MOI. Cell proliferation assay showed that the amount of BrdU incorporated into ZNF403knockdown cells was-50%less compared to that of the control in both Hep-2and HEK293cells at the same time point, implicating that knockdown of ZNF403significantly suppressed cell proliferation. The inhibited cell proliferation by ZNF403knockdown was further supported and validated by tumor graft in nude mice. In addition, soft agar assay indicated that knockdown of ZNF403expression substantially impaired the anchorage-independent growth of Hep-2cells. Furthermore, wound-healing assay demonstrated that the ZNF403knockdown group exhibited an obvious reduction in migration ability as compared to the control groups.In summary, these findings revealed for the first time that ZNF403knockdown remarkably suppressed cell proliferation both in vitro and in vivo, as well as significantly reduced malignancy of laryngeal tumor Hep-2cells.[Function and mechanism analysis of ZNF403knockdown in cell cycle progressionAccording to the cell-cycle associated putative motifs in ZNF403using the software Eukaryotic Linear Motif Resource for Functional site in Proteins, we investigated the effect of ZNF403knockdown on cell cycle progression to elucidate the mechanism of ZNF403in cell proliferation. We showed by flow cytometric analysis of cellular DNA contents using propidium iodide that reduced expression of ZNF403led to the accumulation of cells at G2/M phase, suggesting that down-regulation of ZNF403promotes G2/M arrest in a dose-dependent manner. Moreover, cell cycle analysis at different time point (day2, day4, day6) demonstrated that the impact of ZNF403knockdown on cell cycle progression was not transient, but stable G2/M arrest. Additionally, BrdU was used to illustrate the role of ZNF403in DNA synthesis. To further decipher the mechanism underlying the G2/M cell cycle arrest by ZNF403knockdown, we compared expression level of84cell-cycle genes between ZNF403knockdown group and control group by Human Cell Cycle Profiler realtime PCR Array. Interestingly, the knockdown of ZNF403significantly increased the expression levels of p21, skp2, CDK5R1and CDKN2B, whereas repressed the expression of ATM, MCM2and MRE11A in both Hep-2and HEK293cells. Among them, the changes of P21, MCM2, ATM and MRE11A were further confirmed by western blot analysis.Altogether, these results suggest that the inhibited cell proliferation induced by ZNF403knockdown may due to the G2/M arrest which delayed progress towards mitosis and provide a new insight into the function of ZNF403in regulating the G2/M cell-cycle transition. [cloning and identification of the promoter of ZNF403and analysis of its transcription binding sites]Several bioinformatics programs were utilized to predict the candidate promoter region. Based on the bioinformatics analysis of CpG island, transcription start sites and candidate promoter location by CpGplot, Methprimer, FirstEF, Gene2promoter and NNPP, we selected the1646bps located at (-1465,+199) as the candidate promoter of ZNF403and constructed a series of promoter deletions within luciferase reportor vector (pGL3-Basic). We next performed dual luciferase assays and demonstrated that the core promoter was located at (-926～-682).Next, AHR was predicted to be a transcription factor by Matlnspector V2.2, TESS and ECR. Furthermore, combination of AHR ligand TCDD’s treatment and luciferase activity assay implied the induction of ZNF403by TCDD may be mediated by the transcription regulation of the AHR binding site within its promoter. This finding provides important support that ZNF403is a downstream gene regulated by AHR and also suggests its essential role in pollutant-related tumorigenesis.