Preliminary Functional Studies Of Zinc Finger Protein 307 In The Development Of Hepatocellular Carcinoma

Background and purposeHepatocellular carcinoma（HCC）is one of the most prevalent malignant tumors and the third leading cause of cancer-death in the world.China is the country with the highest incidence of liver cancer worldwide.It is estimated that about 782,500 new liver cancer cases and 745,500 deaths occurred worldwide during 2012,with China alone accounting for about 50%of the total number of cases and deaths.In recent years,although the treatment of liver cancer updated（including surgery,liver transplantation,radiation therapy,interventional therapy,radiofrequency ablation,targeted therapy etc.）,but the overall survival of HCC did not significantly improve as recurrence and metastasis of HCC is still the main prognosis factor.Therefore,elucidating the mechanism of invasion and metastasis of liver cancer,finding the molecular markers of invasion and metastasis of HCC,and discovering new targets of prevention and treatment are the important contents of the study of liver cancer.Zinc finger family of proteins is one of the most common families of transcription factors in eukaryotic cells and has more than 3000 members in the human genome.The functions of zinc finger proteins are of great diversity,including DNA recognition,RNA packaging,transcri-ptional activation,regulation of apoptosis,and so on.Because of their multiple functions,some zinc finger proteins are identified as tumor suppressors or oncogenes in the development of tumor.Zinc finger protein 307（ZNF307）,also known as ZKSCAN4（zinc finger with KRAB and SCAN domains 4）,ZSCAN36 and ZNF427,was originally obtained from the human embryonic heart library PCR cDNA as a template and amplified by PCR.The ZNF307 cDNA sequence is distributed in a sequence of the NT₀07592 genome located on chromosome 6 with a 1638 bp full open reading frame encoding 546 amino acids.ZNF307,as one of the important members of zinc finger protein transcription factor,may be closely related to cell proliferation,apoptosis,tumor development and so on.Jing Li et al.Found that in HEK-293 cells ZNF307 suppressed the transcriptional activity of p53 and p21 by up-regulating mRNA levels of MDM2（p53-binding protein MDM2）and EP300（ElA binding protein p300）.However,whether it has the function of transcriptional inhibition in vivo and what function it plays in the occurrence and development of HCC are largely unknown.Therefore,the purpose of this study is to explore the role of ZNF307 in the development and progression of HCC and to provide a theoretical basis for the treatment of HCC.Methods1.The expression of ZNF307 in HCC cells and tissue samples was detected by fluorescence quantitative PCR The mRNA expression of ZNF307 was detected by fluorescence quantitativePCR in normal liver cell L02 and HCC cells MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be17404.The mRNA expression of ZNF307 in 33 HCC tissue samples and their paired normal liver samples were detected by fluorescence quantitative PCR.2.The expression of ZNF307 in HCC cells and tissue samples was detected by Western blotThe expression of ZNF307 was detected by Western blot in normal liver cell L02 and HCC cells MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be17404.The expression of ZNF307 in 4 HCC tissue samples and their paired normal liver samples were detected by Western blot.3.Establishing the stable ZNF307 knockdown cell lines and verifying the transfection efficiencyThe HCC cells with relatively high expression of ZNF307 were transfected with shRNA containing ZNF307 gene interference fragment from Shanghai Gemma Co.Ltd.,and the vector containing empty vector was used as control.Fluorescence Quantitative PCR and Western blot were used to detect the interference efficiency of ZNF307.4.Establishing the stable ZNF307 overexpressing cell lines and verifying the transfection efficiencyThe HCC cells with relatively low expression of ZNF307 were transfected with Lentivirus virus vector containing ZNF307 gene fragment from Shanghai Gemma Co.Ltd.,and the vector containing empty vector was used as control.Fluorescence quantitative PCR and Western blot were used to detect the overexpression efficiency of ZNF307.5.The effect on biological behavior in vitro of hepatocellular carcinoma cells after ZNF307 interferenceThe effects of ZNF307:interference on the proliferation,migration,invasion and apoptosis of hepatocellular carcinoma cells were detected by plate cloning,CCK-8,scratch test,in vitro invasion assay and flow cytometry.6.The effect on biological behavior in vitro of hepatocellular carcinoma cells after ZNF307 overexpressionThe effects of ZNF307 overexpression on the proliferation,migration,invasion and apoptosis of hepatocellular carcinoma cells were detected by plate cloning,CCK-8,scratch test,in vitro invasion assay and flow cytometry.7.The effect on proliferation biological behavior in vivo of hepatocellular carcinoma cells after ZNF307 interference and overexpressionThe stable knockdown ZNF307 HCC cell line MHCC97L and the stable overexpressing ZNF307 HCC cell line Be17402 were injected subcutaneously on the left and right axillary tissues of the nude mice with the mock cell lines respectively.The nude mice were measured every 5 days and analyzed the data in the end.In vivo experiments revealed the effect of ZNF307 silencing and overexpression on the proliferation of HCC cells.8.The expression of apoptosis-related proteins was detected in ZNF307 silencing and overexpressing HCC cells by Western blotWestern blot was used to detect the expression of apoptosis-related proteins（Caspase3,BAX and BCL-2）in ZNF307 silencing and overexpressing HCC cells,and to explore the possible mechanism of ZNF307 on the apoptosis in hepatocellular carcinoma cell lines.Results1.Downregulated expression levels of ZNF307 mRNA and protein in HCC cell linesThe mRNA expression of ZNF307 was detected by fluorescence quantitative PCR in normal liver cell L02 and HCC cells MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be17404.Results showed that the mRNA expression of ZNF307 is highest in normal liver cell L02,the second order from high to low is MHCC97L,HCCLM3,Huh7 and Be17402 HCC cell lines（P<0.05,P<0.05,P<0.05,P<0.05）.The mRNA expression of ZNF307 in normal liver cell L02 and HCC cell lines QGY7701,QGY7703,Be17404 were not significantly different.It is suggested that ZNF307 gene is generally downregulated expression in hepatocellular carcinoma cell lines.The expression of ZNF307 was detected by Western blot in normal liver cell L02 and HCC cells MHCC97L,HCCLM3,Huh7,QGY7701,QGY7703,Be17402,Be17404.Results showed that the expression of ZNF307 is highest in normal liver cell L02,the second order from high to low is MHCC97L,HCCLM3,Huh7 and Be17402 HCC cell lines.The expression of ZNF307 has no significantly different between normal liver cell L02 and HCC cell lines QGY7701,QGY7703,Be17404.It is suggested that ZNF307 was generally downregulated expression in hepatocellular carcinoma cell lines.Western blot results of ZNF307 protein expression level was in accordance with Q-PCR results of mRNA expression level2.Downregulated expression levels of ZNF307 mRNA and protein in HCC tissuesThe mRNA expression of ZNF307 in 33 HCC tissue samples and their paired normal liver samples were detected by fluorescence quantitative PCR.The results showed that ZNF307 expression in hepatocellular carcinoma samples was lower than that in adjacent normal liver tissues（Z=-4.404,P<0.001）.ZNF307 was down-regulated in hepatocellular carcinoma tissues in transcription level.The expression of ZNF307 in 4 HCC tissue samples and their paired normal liver samples were detected by Western blot.The results show that ZNF307 expression in hepatocellular carcinoma samples was lower than that in adjacent normal liver tissues.ZNF307 was down-regulated in hepatocellular carcinoma tissues in translation level.3.The effect on biological behavior in vitro of hepatocellular carcinoma cells after ZNF307 interference（1）Establishing the stable ZNF307 knockdown cell lines and verifying the transfection efficiency:The HCC cells with relatively high expression of ZNF307 were transfected with shRNA containing ZNF307 gene interference fragment.Results showed that the expression of ZNF307 in the interference group was significantly lower than that in the control group by Q-PCR and Western blot（MHCC97L:t=69.640,P<0.001;QGY7701:t=43.725,P<0.001）.（2）ZNF307 knockdown promoted proliferation of HCC cell lines in vitro:CCK-8 test,plate cloning experiments showed that compared with the control group,ZNF307 knockdown promoted in vitro proliferation ability（MHCC97L:F=62.066,P<0.001;QGY7701:F=10.525,P<0.001）and clone ability（MHCC97L:t=7.252,P<0.05;QGY7701:t=10.713,P<0.001）.（3）ZNF307 knockdown promoted invasion of HCC cell lines in vitro:the matrigel invasion assay showed that compared with the control group,ZNF307 knockdown promoted in vitro invasion ability（MHCC97L:t=13.976,P<0.001;QGY7701:t=25.652,P<0.001）.（4）ZNF307 knockdown promoted migration of HCC cell lines in vitro:wound healing assays showed that compared with the control group,ZNF307 knockdown promoted in vitro migration ability（MHCC97L:t=22.979,P<0.001;QGY7701:t=19.489,P<0.001）.（5）ZNF307 knockdown decreased the proportion of early and late apoptosis of HCC cell lines in vitro:flow cytometry showed that compared with the control group,ZNF307 knockdown significantly decreased the proportion of early and late apoptotic cells（MHCC97L:t=8.414,P<0.05;QGY7701:t=47.369,P<0.001）,it is suggested that ZNF307 knockdown suppressed the apoptosis of HCC cell lines.4.The effect on biological behavior in vitro of hepatocellular carcinoma cells after ZNF307 overexpression（1）Establishing the stable ZNF307 overexpressing cell lines and verifying the transfection efficiency:The HCC cells with relatively low expression of ZNF307 were transfected with Lentivirus virus vector containing ZNF307 gene fragment.Results showed that the expression of ZNF307 in the overexpressing group was significantly higher than that in the control group by Q-PCR and Western blot（Be17402:t=105.364,P<0.001;HCCLM3:t=126.165,P<0.001）.（2）ZNF307 overexpressing suppressed proliferation of HCC cell lines in vitro:CCK-8 test,plate cloning experiments showed that compared with the control group,ZNF307 overexpressing suppressed in vitro proliferation ability（Be17402:F=27.136,P<0.001;HCCLM3:F=4.541,P<0.05）and clone ability（Be17402:t=16.670,P<0.001;HCCLM3:t=10.169,P<0.05）.（3）ZNF307 overexpressing suppressed invasion of HCC cell lines in vitro:the matrigel invasion assay showed that compared with the control group,ZNF307 overexpressing suppressed in vitro invasion ability（Be17402:t=8.358,P<0.05;HCCLM3:t=24.607,P<0.001）.（4）ZNF307 overexpressing suppressed migration of HCC cell lines in vitro:wound healing assays showed that compared with the control group,ZNF307 overexpressing suppressed vitro migration ability（Be17402:t=19.176,P<0.001;HCCLM3:t=6.031,P<0.05）.（5）ZNF307 overexpressing promoted the proportion of early and late apoptosis of HCC cell lines in vitro:flow cytometry showed that compared with the control group,ZNF307 overexpressing significantly increased the proportion of early and late apoptotic cells（Be17402:t=7.790,P<0.05;HCCLM3:t=17.451,P<0.001）,it is suggested that ZNF307 overexpressing promoted the apoptosis of HCC cell lines.5.The effect on proliferation biological behavior in vivo of hepatocellular carcinoma cells after ZNF307 interference and overexpressionThe results showed that,compared with the control group,ZNF307 knockdown promoted in vivo proliferation ability（F=31.378,P<0.05）.On the other hand,ZNF307 overexpressing suppressed in vivo proliferation ability（F=33.629,P<0.05）.6.The effect on the expression of apoptosis-related proteins in hepatocellular carcinoma cells after ZNF307 interference and overexpressionWestern blot was used to detect the expression of apoptosis-related proteins（Caspase3,BAX and BCL-2）in ZNF307 silencing HCC cells,and found that the expression of BCL-2 was upregulated and the expression of Caspase3 and BAX were downregulated in ZNF307 silencing HCC cells,compared with the controls.On the other hand,overexpression of ZNF307 led to the fact that expression of Caspase3 and BAX were increased and the expression of BCL-2 was decreased significantly.Therefore,the results indicate that ZNF307 may serve as a tumor suppressor and inhibits cell proliferation of HCC cells via regulating the apoptosis-related proteins.Conclusion1.The expression of ZNF307 in hepatocellular carcinoma cells/tissues is significantly lower than that in normal human liver cells/tissues.2.ZNF307 inhibits proliferation,invasion,migration and promotes apoptosis of hepatocellular carcinoma.3.ZNF307 may inhibit the proliferation of hepatocellular carcinoma cells via regulating the apoptosis-related proteins.