Preliminary Functional Studies Of Gene ZNF 217 In Progression Of Ovarian Cancer

BACKGROUND&OBJECTIVEOvarian cancer is one of the three female main reproductive system cancers.The five-year survival rate is only 25%-30%in patients in old age.The morbility of ovarian cancer in china is increasing fast during these several years,and the patients' age tends to younger.With the development of cancer genetics and molecular biology technology,pathogenesis of ovarian cancer have been extensively and studied. Recognizing that the abnormal exchange of genetic material is the main cause of tumor accurrence.The incidence of tumor is based on the activation of oncogenes and tumor suppressor gene inactivation.The same genetic abnormality exists in the same types of tumor cells which suggests that these anomalities are a major cause of cancer.The occurrence and development of ovarian cancer refers to multi -abnormalgenegene.It is a hot topic looking for chromosomal abnormalities associated with cancer and identification of disease genes in recent years. Chromosome abnormalities are widely found in ovarian cancer genome by hybridization technique comparative,Gene amplification has higher probability in the chromosome 20,particularly frequent sites 20q13.2,which contains the two genes STK15 gene and ZNF217. ZNF217 gene located in chromosome 20q13.2 is a newly cloned cancer gene in the 20th chromosome,which codes Krupple like transcription factor and belongs to zinc finger protein family.ZNF217 gene,as a transcriptional regulatory factor,may be involved in tumor occurrence and development.An increasing number of studies show that at present,Zinc finger protein family members play an important role in the development of a variety of cancers.Kate G.R.Quinlan,etc.proved ZNF217 restrains the role of transcription through mutual reaction between PXDLS sequences(At the combination of gullies of the C-terminal-binding protein-binding domain),RRT sequences(Combining in domain of the surface of the ditch of the C-terminal-binding protein nucleotide-binding domain)and the C-terminal-binding protein.With the increase of copy number of ZNF217,gene expression changes accordingly,for example,inhibiting the expression of tumor suppressor gene promoter can lead to the occurrence of tumor.Authors speculate ZNF217 may be sequence-specific DNA-binding protein and silence a certain gene through a combination of C-terminal-binding protein.Peixiang Li etc.think ZNF217 plays the following main roles in the occurrence of tumors:Lowing the expression level of certain tumor suppressor,raising the expression level of oncogenes,inducing anchorage- independence and serum - independence,activating autocrine of growth-promoting Factor,and for the first time found that the difference expression of ZNF217 in different cells has a relation with the status of p53 and pRB. We found a variety of initiation factor combinate ZNF217 and C-terminal-binding protein,these initiation factors are activated without ZNF217.ZNF217 gene in esophageal squamous cell carcinoma,gastric cancer,prostate cancer, colorectal cancer study,is believed to be involved in occurrence and development process of cancer.ZNF217 gene in breast cancer research is relatively clear,which is one of the two promoting factors for breast cancer.Tanner etc.have detected ZNF217 gene amplification and higher expression in ovarian cancer patients.However,the structural change is limited to the study of this gene,papers related to its current function are not seen.This study focused on the role of ZNF217 gene in the occurrence and development process of ovarian cancer,and laid the theoretical foundation of the mechanism for ovarian cancer.METHODS1.Detection of ZNF217 amplification in ovarian cancer tissue and ovarian cancer cell lines by Fluorescence in situ hybridizationZNF217 gene was detected in the tissue of ovrian cystadenocarcinoma of different stages,serous cystadenoma and normal tissue of ovarian.The ZNF217 amplification in HO-8910,SKOV3,OVCa-R3 ovarian cystadenocarcinoma cells is also detected.2.Detection of expression of ZNF217 in ovarian cancer cell lines and in ovarian cancer tissue by RT-PCRZNF217 gene was detected by RT-PCR in ovarian cancer cell lines and in ovarian cancer tissue.And cell lines with high expression of ZNF217 gene were screened for the follow-up of RNA interference experiment. 3.Effect of ZNF217 gene silence on the biological characteristics of ovarian cancer ZNF217 gene interference fragment,recombinant Plasmid vector pGenesil/ ZNF217shRNA is designed by genesil company,Using cationic lipids Lipofectaming _TM2000,the pGenesil/217shRNA was transfected into epithelial ovarian cancer cell line HO-8910,and use of negative space vector as the control.After resistance monoclonal selected with G - 418,a strong fluorescent clones expanding culture Quantitative is picked,and detected by RT-PCR and Western blot.Expression of ZNF217 gene after interference was detected by MTT and fiat colony rate.The invasive of silent ZNF217 gene expression was detected by invasion and sporting ability in vitro.4.Effect of ZNF217 gene silence on the ovarian cancer cell proliferation in vivo Cells proliferation ability of ovarian cancer was detect by MTT,after interference.5.Analysis of the effect of ZNF217 gene on cell signal transduct of ovarian cancer mechanism by gene chip technologyDifferentially expressed genes expression pedigree were detect after ZNF217 silence gene expression by Oligonucleotide mieroarray of Affymetrix company.And we analysis these genes by Gene Ontology,MILANO,Panther,Genomatix software and GeneCLIP.We can draw ZNF217 signal pathway map in the development of ovarian cancer.Genes expression were detected by Quantitative PCR. 6.STATISTICAL ANALYSISThese data were analyzed by SPSS 13.0 software,FISH and RT-PCR were based on two independent samples x² test and R×C lists x² test analysis,the independent samples t-test was used to analyze the datas which refer to movement experiment invasion experiment colony formation rate experiment weight of tumor in situ, subcutaneous tumor experiment and proliferation experiments in vitro were assessed by means of the repeated- measures analysis of variance.RESULT1.Detection of ZNF217 amplification in ovarian cancer tissu and ovarian cancer cell lines by Fluorescence in situ hybridization In 11 eases of stageâ… ovarian serous cystadenocarcinoma,3 cases had over 2 red fluorescen signals,and stageâ…¢-â…£(12 cases)had nine cases,Ovarian serous cystadenoma(10 cases)had basically two red fluorescent signal,1 patient had three red fluorescen signal.In 12 normal ovarian tissue every had 2 fluorescen signals.The amplication of ZNF217 was gained in 12 cases of ovarian cancers(52.17%), including 9(75.00%)advanced ovarian cancer,and 3(25%)early ovarian cancer, (x²=5.239,p = 0.039).There is only 1 ease in ovarian benign tumor,which may be an accident Significant difference between benign tumors and normal tissues was detected(x²=12.682,p=0.002).The amplication of ZNF217 exsisted in HO-8910,SKOV3,OVCa-R3 three ovrian serous cystadenocarcinoma cell lines.2.The gene ZNF217 was detection in ovarian cancer cell lines and in ovarian cystadenocarcinoma by RT-PCRZNF217 gene is expressed highly in human ovarian carcinoma cell line HO-8910.About ZNF217mRNA,Cancer had a significant between from benign tumors and normal tissues(x²=13.705,p=0.001).13 ZNF217 high expression ovarian cancers,Stageâ… ovarian cancer accounts for 23.07%(3/13)Phaseâ…¢-â…£76.03%(10/13).Advanced ovarian cancer and early were significantly different (x²=6.418,p=0.027).These showed expressions of ZNF217 was higher in ovarian carcinoma cells than benign tumor and normal ovarian tissue.The expressions of ZNF217 in the advanced ovarian cancer were higher than the early stage.RT-PCR results show the ZNF217 gene is expressed highly in human ovarian carcinoma cell line HO-8910.RNAi interference can be used for follow-up experiments.3.Role of RNAi of ZNF217 gene in cells of HO-8910Vector pGenesil/ZNF2171shRNA and pGenesil/ZNF2172shRNA were constructed by PGenesil Plasmid vector system in view of the target gene ZNF217.It was confirmed by sequencing results that pGenesil pGenesil/ ZNF2171shRNA and pGenesil/ ZNF2172 shRNAcells transfected cell HO-8910.The pGenesil/ ZNF2171shRNA interference fragment was in the follow-up experiments. Interference clips and negative control HO-8910 cells selected with G - 418 get resistance clones.Quantitative RT-PCR and Western Blot results showed ZNF217 has the most obvious reduction of ZNF217 in interference cloning 2(named HO-8910/ZNF217^-),the rate of interference amounted to 86%.4.Effect of ZNF217 gene silence on the biological characteristics of ovarian cancer ZNF217 cell proliferation in vitro was observed after gene silence,it is showed that compared with HO-8910/HK and HO-8910,HO-8910/ZNF217 proliferation is significantly reduced.Three groups were significantly different(F=19.115,p=0.000). Flat cloning experiment results also showed HO-8910/ZNF217^-cells in a single cell proliferation significantly was lower than other groups.The difference was statistically significant(t=4.354,p=0.012).These results revealed that tumor growth was significantly inhibited in vitro after reduction of ZNF217 gene expression. Invasion chamber assay in vitro detected the changes in the ability of cell invasion. After ZNF217 gene silence,the invasion of HO-8910/ZNF217^- was significantly lower than the HO-8910/HK(t=6.613;p=0.000).It showed ZNF217 silence significantly inhibited the invasive ability of ovarian cancer cells in vitro. Movement experiment in vitro detected the changes in the ability of cell.After ZNF217 gene silence,the movement of HO-8910/ZNF217^- was significantly lower than the HO-8910/HK(t=13.013;p=0.000).It showed that ZNF217 silence significantly inhibited the movement of ovarian cancer cells in vitro. Through visualization animal model of ovarian cancer,we observed the effects of ZNF217 gene silence on the proliferation and metastasis of ovarian cancer.We inoculated varian cancer cells in nude mice subcutaneously.It is showed that the proliferation of tumor cells in vivo after 21 days for observation which was significantly inhibited after ZNF217 gene silence.We found the proliferation difference from the 15th day to 21th day,is three times.5.Exploring the role of ZNF217 gene in ovarian cancer proliferation,invasion,the role of metastesis by chip technologyThrough analysis of gene expression of HO-8910/ZNF217-and HO-8910/HK before and after interference by the Human Genome U₁33plus2 synthetic oligonucleotide microarray in situ prepared by Affymetrix,we got the gene signaling pathway.Cluster analysis revealed three main signal cluster network,at the core of ZNF217 in primary position.After silencing ZNF217,we can drawed 94 genes which are the tumor apoptosis,invasion and metastasis gene.MMP-24 gene which lowered 32 times,this gene in normal and tumor tissues were expressed,frequently in various tumor detection of the gene amplification,but no ovarian cancer related literature.ZNF217 in primary combined with the downturn after the silent gene-mediated signaling pathway in ovarian cancer analysis,speculate MMP-24 gene might be involved in ovarian cancer mechanism of the new gene,specific mechanisms require further study confirmed.CONCLUSION1.ZNF217 gene is a promoter which play a roles in the proliferation,invasion,and movement of ovarian cancer.ZNF217 may be the target gene for ovarian cancer therapy.2.ZNF217 closely cooperates with the MMP-24.MMP-24 may be an new gene that play a role in the ovarian cancer progression.