Effect Of Lentivirus-Mediated RNAi DDAH2 Gene Silence On Biological Behaviors Of Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is the predominant primary liver cancer in many countries and the sixth most common cancer worldwide.In China,HCC is the second most common cause of cancer-related death.Though surgical ablation and liver transplantation are the effective measures for treating HCC,the effect is dissatisfactory.Metastasis and recurrence of HCC are the maximal barrier influencing therapeutic effect.Deepening our understanding of the tumor's biology characteristics,exploring the development intrinsic mechanism,and seeking for effective early diagnosis and treatment methods to improve the curative effect of HCC can improve the prognosis of patients with HCC.Invasion and metastasis is an important biological behavior of malignant tumors.Liver tumors display marked vascular abnormalities.Angiogenesis may promote HCC growth and progression.Dimethylarginine dimethylaminohydrolase 2(DDAH2)plays a central role in mediating tumor angiogenesis.DDAH2,enzyme which metabolizes the endogenous NOS inhibitor asymmetric dimethylarginine(ADMA),reduces tissue ADMA levels and enhances angiogenesis.Previous studies demonstrated that DDAH2 expression played important roles in the regulation of intratumoral angiogenesis and was associated with neovascularization,malignant potential,and unfavorable prognosis of patients with breast carcinoma,lung adenocarcinoma and prostatic cancer.However,no study was conducted to focus on the expression pattern in tumor cells and potential role of DDAH2 in HCC.Therefore,this research combines clinical data analysis,RNA interference silencing genes,and in vivo and vitro experiment,observes the DDAH2 gene's role in HCC through patients,molecular and cellular,and discusses its mechanism,to provide a new gene target for the diagnosis and treatment of HCC.Outline:Chapter 1:To observe the expression differences of DDAH2 gene in HCC tissue and adjacent non-tumorous liver tissue,then to discuss the relationship of expression level with tumor differentiation,invasion and metastasis ability.Chapter 2:To construct the lentiviral vector which targets at interfering DDAH2 gene expression,then to verify the interference effect for the next function experiment;Chapter 3:To use the lentiviral vector which targets at interfering DDAH2 gene expression to infect SMMC-7721 cells,then to observe the invasion and metastasis abilities change.Chapter 1 Expression and Its Significance of DDAH2 in Human Hepatocellular carcinoma Tissue and CellObjective:To observe the expression differences of DDAH2 gene in HCC tissue and adjacent non-tumorous liver tissue,to discuss the relationship of expression level and tumor differentiation,invasion and metastasis ability,and to observe the expression of DDAH2 gene in SMMC-7721 cells.Methods:By using immnnohistochemical method to detect DDAH2 expression in 40 cases of HCC tissue and 40 cases of adjacent non-tumorous liver tissues,to collect HCC patients clinical data,to analyze the relationship between DDAH2 gene expression and HCC patients clinical and pathological features.Results:The positive rate of DDAH2 protein expression in HCC tissue is obviously higher than that in the adjacent non-tumorous liver tissues(P<0.001).HCC DDAH2 protein expression was correlated with microvascular invasion(P<0.05),while has no significant correlation with gender,age,TNM stage,the differentiation degree,Child-Pugh degree(P>0.05).DDAH2 has expressions in SMMC-7721 cell.Conclusion:(1)DDAH2 expression in HCC tissues were expressed significantly higher,and increased with the rise of microvascular invasion;(2)DDAH2 expression in SMMC-7721 cells is higher,so choose SMMC-7721 cells for next experiment.Chapter 2 Production,Packaging and Virus Titer Detection of Lentiviral Vector of DDAH2-shRNAObjective:To screen effective DDAH2-siRNA sequence,construct recombinant lentiviral vector of DDAH2-shRNA,and detect the titer of virus then package virus.Methods:Design the siRNA sequence according to the DDAH2 target gene sequence;assess transfection efficiency by observing the expression of reporter gene after the SMMC-7721 cell infection by DDAH2-shRNA lentiviral vector;evaluate interference efficiency by detecting the expression of DDAH2 using Real-time PCR;construct satisfactory lentiviral vector and packaging.Results:Three RNAi lentivirus expression vectors targeting to three various sites of DDAH2 gene were produced,and the sequence and correct site of ds oligo inserted were confirmed by PCR and sequencing assay;The lentivirus were packaged in 293T cells with high titer;Small hairpin RNA(shRNA)targeting vasohibin-1 was transfected into SMMC-7721 cells.The efficiency of DDAH2 RNAi sequence against the expression of DDAH2 was detected by Real-time PCR and western blot.Real-time PCR showed that the relative mRNA levels in DDAH2 RNAi-1,2,3,group was 32.1%,65.6%and 48.3%,compared to the negative control group.All the three RNAi significantly reduced the DDAH2 mRNA(P<0.05),and the DDAH2 RNAi 1 showed strongest inhibition.Western blot showed that the protein level of DDAH2 was down-regulation in the DDAH2 RNAi,with strongest down-regulation in the DDAH2 RNAi 1 group(P<0.05).Real-time PCR and western blot suggested that DDAH2 RNAi 1 showed strongest inhibition of the expression of DDAH2 compared with DDAH2 RNAi 2,3 DDAH2 RNAi 1 was choosed for the following experiments to investigate the role of DDAH2 in the invasion of HCC cell lines.Conclusion:DDAH2-shRNA Ientiviral vector was constructed successfullly,which can effectively transfect SMMC-7721 cells,and effectively down-regulate SMMC-7721 cell DDAH2 gene expression.Chapter 3 Effects of DDAH2 Gene Expression Inhibition by RNAi on HCC Cell Biological CharacteristicsObjective:To study the effects of DDAH2 targeted inhibition on HCC cell proliferation,migration ability and a.ngiogenesis.Methods:SMMC-7721 cells are divided into three groups.The experimental group and the control group were transfected by DDAH2-shRNA lentiviral vector and shRNA negative control lentiviral vector,and nothing is done with blank group.By using MTT experiment detect cell proliferation activity,and by using scratch wound healing assay detect cell migration vitality.To investigate the possible mechanism of DDAH2 involved in the procession of angiogenesis,the expression of VEGF in SMMC-7721 after DDAH2 RNAi transfection was tested by Real-time PCR and western blot.Results:Role of DDAH2 RNAi on proliferation of SMMC-7721 cell:1)DDAH2 RNAi sequence could decrease the SMMC-7721 cell proliferation.A MMT assay revealed that treatment with DDAH2 RNAi did change the number of viable cells,compared with the control groups(P<0.05).2)vasohibin-1 RNAi sequence could change the migration of SMMC-7721 cell:Scratch wound healing results showed that the wound distance 24h and 48h after scratching in negative control cells,The migrating distances of DDAH2 RNAi cells was significantly shorter than those of NC RNAi cells and negative control cells(P<0.05).3)DDAH2 RNAi sequence down-regulated the expression of VEGF:Real-time PCR suggested that the expression of VEGF was significant lower in DDAH2 RNAi group than in the control groups(p<0.05).Western blot results further confirmed that the expression of VEGF was down regulated in the DDAH2 RNAi group.Conclusion:The silence of DDAH2 can inhibit the SMMC-7721 cell proliferation and migration activity.Above all,the expression of VEGF downregulation,which suggested that DDAH2 may be involved in angiogenesis of hepatocellular carcinoma cells by regulating the expression of VEGF.