Research On The Effect Of ZNF217 In Hepatocellular Carcinoma And Its Potential Mechanism

Liver cancer is one of the most prevalent malignant tumors in China,accounting for third of all malignant tumor incidence,and its mortality rate accounts for second of all malignant tumor related deaths.China has a huge population of hepatitis B,and many patients fail to get early diagnosis and treatment.Once found,most tumors have been in the middle and late stages.Although surgical treatment together with other comprehensive treatments has significantly improved the treatment effect,for the patients fail to carry out surgical resection of the tumor,there is still a lack of effective treatments.In recent years,to intervene hepatocellular tumor from proliferation and metastasis via various ways becomes a research direction and focus.The occurrence of liver cancer is related to the change of gene expression,and the abnormal proliferation and metastasis are also regulated by gene expression.Zinc finger protein 217(ZNF217)is a member of the zinc finger protein family,whose zinc finger structure is confirmed to be effective in gene transcription regulation.In recent years,many studies found that zinc finger protein 217 is up-regulated in breast cancer,ovarian cancer,colorectal cancer,gastric cancer compared with the matched normal tissues.Besides,epithelial mesenchymal transformation(EMT)is proved to promote the invasion and metastasis of tumor,suggesting that the zinc finger protein 217 may be linked to liver cancer occurrence and development.Part I ZNF217 is up-regulated in hepatocellular carcinoma,promotes tumor cells proliferation and represses apoptosis.Aims:ZNF217 is up-regulated in breast cancer,ovarian cancer,colorectal cancer,gastric cancer compared with the matched normal tissues.Expression of ZNF217 in hepatocellular carcinoma,promotion of cell proliferation and repression of cell apoptosis are confirmed in this paper.Methods:Expression of ZNF217 protein level in 22 pairs of liver cancer and matched adjacent normal liver tissues were analyzed by immuno-histochemistry.Detection of mRNA level and protein level of ZNF217 in hepatocellular cell lines SK-HEP-1,Hep-G2,Huh-7,PLC/PRF/5 and normal liver cell line L-02 were performed.Knock down of ZNF217 model was established by using small interfering RNA,and the over-expression model was established by adding recombinant ZNF217 via adenovirus vector.Cell proliferation activity was evaluated by CCK-8 assay and EdU assay before and after treatment.The effect of ZNF217 on cell apoptosis and cell cycle were analyzed by flow cytometry.The expression of apoptosis related protein and cell cycle related protein were detected by blot Western method,and the potential mechanism were discussed.Results:Immuno-histochemistry of 22 clinical tissue samples indicates that 16 cases(73%)of patients have a higher expression of ZNF217 in tumor tissues compared with the matched adjacent normal liver tissues,and 4 cases(18%)of patients have no significant difference between tumor and normal tissues,2 cases of patients(9%)have a higher expression of ZNF217 in adjacent normal liver tissues.ZNF217 was richer in four kinds of liver cancer cell lines,Hep-G2,Huh-7,SK-HEP-1,PLC/PRF/5 than the normal liver cell line L-02 both in mRNA and protein levels.Cell proliferation activity decreased significantly(p=0.0052)in small interfering RNA treatment group compared with the control group in CCK-8 assay and EdU experiments.More cells in G1 phase arrest were detected via cytometry analysis,with increasing expression of CyclinD1 and CDK4.Besides,cyclinB1 expression decreased compared with negative control.Increasing proportion of apoptotic cells were detected via cytometry analysis,with richer expression of Bax and cleaved caspase-3.Cell proliferation activity increased significantly(p=0.0007)in adenovirus-ZNF217 treated group compared with the control group in CCK-8 assay and EdU experiments.Blockage in G1 phase relief were detected via cytometry analysis,with decreasing expression of CyclinD1 and CDK4.Besides,cyclinB1 expression increased compared with negative control.Decreasing proportion of apoptotic cells were detected via cytometry analysis,with lower expression of Bax and cleaved caspase-3.Conclusion:ZNF217 is up-regulated in hepatocellular carcinoma.ZNF217 promotes cell proliferation by relieving G1 phase blockage,and inhibits cell apoptosis potentially via down-regulated caspase-3 signaling pathway.Part II ZNF217 triggers cell migration in hepatocellular carcinoma via epithelial-mesenchymal transitionAims:ZNF217 significantly promotes cell proliferation and relieve G1 phase blockage.In previous study on breast cancer,ZNF217 is involved in the process of epithelial mesenchymal transition(EMT),by which the ability of cell migration,invasion and distant metastasis is greatly enhanced.This study was to investigate the enhancement of cell migration triggered by ZNF217via epithelial mesenchymal transition in hepatocellular carcinoma.Methods:Knock down model of ZNF217 was established by using small interfering RNA,and the recombinant ZNF217 added via adenovirus vector was established as the over-expression model of ZNF217.Cell migration ability were evaluated by wound-healing assay and Transwell assay before and after treatment.Western Blot method was used to detect the epithelial and mesenchymal marker proteins in epithelial mesenchymal transition,and further explored its potential mechanism.Results:In ZNF217 knock-down group,wound healing assay suggested that,18 hours after treatment with small interfering RNA,the remaining scratch width percentage in experimental group was significantly wider than that in the control group(P<0.0001);Transwell assays indicated that less cells penetrated the chamber in si-RNA treated group(P<0.0001).In contrast,the remaining scratch width percentage in over-expression group was significantly narrower than that in the control group(P<0.0001).Besides,cell percentage penetrated the chamber in adenovirus-ZNF217 group increased significantly compared with the control group(P<0.0001).Conclusion:This study showed that ZNF217 can promote cell migration in hepatocellular carcinoma,where epithelial mesenchymal transition may be an important underlying during the process.