ZNF498 Selectively Regulates P53-mediated Apoptosis To Promote The Tumorigenesis And Development Of Hepatocellular Carcinoma

Objective: ZNF498 is one of the largest transcriptional regulators in mammalians,So far,there have been no reports on the biological function and mechanism of ZNF498.And the function and signaling pathway of ZNF498 are unclear.We have confirmed the function and mechanism of Apak,another member of the KZNF family.We found that Apak inhibits p53 activity as transcription factor and transcriptional regulator.Under normal conditions,Apak interacts with p53 by recruiting KAP1 and histone deacetylase,inhibits the transcipitional activity of p53 and apoptosis.In a variety of stress,Apak is separated from p53,promotes p53 activity and induces cell apoptosis.To further understand the function and mechanism of other members of the KZNF family,We screened the members of the KZNF family and found out the ZNF498,could negatively regulate p53 activity.Because ZNF498 had two transcriptional variants,the ZNF498 transcipt variant1(encoding 544 amino acids)and the ZNF498 transcipt variant 2(encoding 465 amino acids),and their functional domain were different,we constructed two expression plasmids of ZNF498(Myc-ZNF498-1 and Myc-ZNF498-2).Our previous studies have shown that ZNF498 was highly expressed in liver cancer tissues and could be associated with the tumorigenesis.As one of the five malignant tumors,liver cancer has a high morbidity and mortality,which is a disease that seriously endangers the life and health of our people.Therefore,the study of liver cancer has become an urgent matter.In molecular and cell levels,threrfore,we will discuss how two isoforms of ZNF498 regulate p53 activity and participate in the development and carcinogenesis of hepatocellular carcinoma.These studies will contribute to elucidate the function of ZNF498 and the mechanism of hepatocellular carcinoma tumorigenesis and progression.Methods: Double luciferase activity assay was used to detect the effect of ZNF498 on p53 transcriptional activity in overexpressed ZNF498 Hep G2 cells.Western blot and q PCR were respectively performed to investigate the effect of ZNF498 on the protein and RNA levels of p53 target gene in Hep G2 cells with ZNF498 knockdown.Co-immunoprecipitation was used to examine interactions between exogenous ZNF498 and p53,the interaction between endogenous ZNF498 and p53 as well as the interaction between ZNF498 fuctional domain and p53,to confirm that ZNF498 interacts with p53.Western blot was used to detect the effect of ZNF498 on p53 acetylation and phosphorylation sites in Hep G2 cells with ZNF498 knockdown and overexpression,to clarify the effect of ZNF498 on p53 post-translation.Staining with Annexin V was performed to explore apoptosis ratio of the defferent cells(p53-/-and p53+/+)which were transfected with plasmids of control and ZNF498 or sh RNA and stable knockdown of ZNF498 expression,to confirm the effect of ZNF498 on cell apoptosis.Cells with p53 deficiency were transfected with wild type p53 or p53Ser46 A mutation,double luciferase activity assay,staining with Annexin V as well as western blot,were respectively used to explore the effect of ZNF498 on p53 transcipitional activity,on cell apoptosis and on Puma protein levels,to confirm whether ZNF498 inhibits apoptosis depend on p53 phosphorlation on Ser46.Hep G2 cells with stable knockdown of ZNF498 expression were used to detect cell proliferation and tumorigenesis,to explore the role of ZNF498 in the development and carcinogenesis of hepatocellular carcinoma.Western blot was used to detect ZNF498 expression in a variety of liver cancer cell lines,as well as liver cancer tissues and adjacent tissues,to confirm the relevance ZNF498 with liver cancer.Results: The two isoforms of ZNF498 inhibited p53 transcriptional activity.Knockdown of ZNF498 in Hep G2 cells enhanced the protein and m RNA levels of Puma,which was p53 target gene.The two isoforms of ZNF498 suppressed Puma protein levels in Hep3 b cells(p53 deficiency)when p53 was overexpressed.Under the DNA damage,the knockdown and overexpression of ZNF498 in Hep G2 cells,respectively promoted and inhibited the expression of Puma.Endogenous and exogenous ZNF498 interacts with p53,and the interaction site is ZNF498 zinc finger domain.Under the condition of DNA damage,the interaction between ZNF498 and p53 did not change.Under normal conditions and DNA damage,the overexpression of ZNF498 inhibited the phosphorylation level of p53 Ser46,but no effect on modification of other sites of p53 in Hep G2 cells.However,Knockdown of ZNF498 in Hep G2 cells promoted the phosphorylation level of p53 Ser46 under normal conditions and DNA damage.ZNF498 inhibited p53 trandcriptional activity,target gene Puma,and cell apoptosis by inhibiting phosphorylation of p53 Ser46.Stable knockdown and overexpression of ZNF498 in Hep G2 cell lines,respectively inhibits cell proliferation and promotes cell proliferation.Stable knockdown of ZNF498 in Hep G2 cell lines was injected into the back of the nude mice and found that the knockdown of ZNF498 inhibited the development of the tumor.ZNF498 expression was upregulated in human HCC cells,as well as HCC tissues,compared with the adjacent tissues.Conclusions: 1.Under normal conditions and DNA damage,ZNF498 inhibits p53 transcriptional activity and selectively inhibits the expression of Puma,p53apoptosis-related target gene.2.ZNF498 interacts with p53.3.ZNF498 inhibits phosphorylation of p53 Ser46,and has no effects on other p53 modification.4.ZNF498 inhibits p53 transcriptional activity,apoptosis and Puma expression through inhibiting phosphorylation of p53 Ser46.5.ZNF498 is highly expressed in liver cancer cells and tissues,ZNF498 promotes HCC cell proliferation and cell growth in vivo.