The Association Research Of Zinc Finger Protein Apak And Tumor

BackgroundKZNF proteins are transcriptional regulatory factors of mammals, which includeKRAB domains and a plurality of zinc fingers. The zinc finger can combine withspecific gene(about3~4nucleotides), and different zinc finger can bind to differentsequences of DNA. KRAB domain can raise histone methyltransferase enzyme, histonedeacetylase, heterochromatin protein to inhibit transcription. KZNF can be binded to thetarget genes by zinc finger, and then inhibit the transcription of target genes by KRABdomain, to regulate activities of cells. KZNF proteins have the following functions:inhibiting the transcription of primers of RNA polymerase I, II and III; regulatingnucleolus; splicing RNA.Apak belongs to KZNF proteins which is found in our laboratory and encoded byZNF420gene. It can regulate p53negatively. In normal cells, Apak recruits KAP1,HDAC1and ATM kinase to bind with p53, so as to downregulate the level ofacetylation of p53and lead to apoptosis. In the situation of MMS-induced DNA damage,ATM is activated which leads to phosphorylation of the Ser68site of Apak, dissociationApak from p53, p53activation, and apoptosis.ObjectiveTo explore if Apak as a member of KZNF family has other fuctions which do notdepend on p53, if Apak can promote growth and proliferation of cell, if Apak can resultin tumor. Hope to give a new warning molecule for diagnosis and prevention of cancer.MethodsThe experiment takes case-control study, which selected tumor samples of15cases of gastric cancer and20cases of colorectal cancer as the case group, while thenormal tissue next to the same patient’s tumor as the control group. Western blottingwas used to detect the expression of Apak in tumor tissue. H1299cells were culturedand transfected by GFP-C3, GFP-Apak, shRNA-con, shRNA-Apak. Real-time PCR wasused to detect rRNA, flow cytometric for cell cycle, MTT for cells proliferation andChIP for DNA sequences Apak protein binding to.Results1. In this study, selecting tumor samples of15cases of gastric cancer and20cases of colorectal cancer as the case group, while the normal tissue next to the same patient’stumor as the control group. Western blotting was used to detect the expression of Apak.It was found that for the same patient, compared with adjacent normal tissue, level ofApak of tumor tissue decreased and in p53mutant cells, level of Apak protein decreasedmore significantly, indicating Apak has other mechanisms which do not depend on p53.2. H1299cells were cultured and transfected by GFP-C3, GFP-Apak, shRNA-con,shRNA-Apak respectively.48hours after transfection, receiving cells and extractingRNA. Real-time quantitative PCR was used to detect the expression of rRNA andWestern blotting was used to detect the expression of Apak. It was found that Apakprotein can inhibit the synthesis of rRNA.3. H1299cells were cultured and transfected by GFP-C3, GFP-Apak respectively.48hours later the cells were received. Cell cycle was detected by flow cytometry. It wasfound that Apak make most of H1299cells blocked in G1.4. H1299cells were cultured and transfected by GFP-C3, GFP-Apak, shRNA-con,shRNA-Apak respectively.48h after transfection, cells were treat with G418. The celllines of stable expression and stable knockdown Apak were get after2~3weeks. MTTwas used to detect cell proliferation. It was found that Apak can inhibit proliferation ofcells.5. It was found that Apak can bind to the regulatory region of45SpreRNA bychromatin immunoprecipitation (ChIP) and the binding sequence of DNA isTCTTN2-30TTGT.6. Set three experimental groups, each containing two bottles which weretransfected with shRNA-con and shRNA-Apak respectively. Take the first groupwithout any drug as a control group; Add TSA into the second group; Add5’-Aza intothe third group. A period of time later, extracting RNA from the cells. Real-timequantitative PCR was used to detect level of preRNAl. The results showed that Apakcan makes the regulatory region of rRNA methylation.Conclusions1. The level of Apak in tumor tissue decreases, especially in the p53mutant cells.2. Apak can bind to the regulatory region of45SpreRNA.3. Apak can make the regulatory region of rRNA methylation.4. Apak can inhibit the synthesis of rRNA.5. Apak protein can inhibit proliferation of cells and make the cells blocked inthe G1phase.