Human AKT2 Gene Therapy Of Liver Cancer Mediated By SiRNA

Liver cancer is one of the common types of gastrointestinal tumors with poor prognosis metastasis and high mortality in which hepatocellular carcinoma(HCC)accounts for about 90%. Although the different available multi-modalities of therapies,including radical surgical operation,intervention therapy,laser therapy et al,have currently been used in the treatment for liver cancer,the prognosis of liver cancer has not been markedly improved. Therefore, it has an important clinic significance to study on the mechanism of the development and progression of liver cancer and the new strategies for the treatment of liver cancer with the molecular biology. With the development of the basic research on oncological fields, it was found that singal conduction has the focus.It was researched that PI3K/AKT singal conduction takes an important role in the growth,differentiation,angiogenesis,metastasis,proliferation of tumors. AKT2 is the key point in the PI3K/AKT singal conduction.It is an important factor. many tumors have the over expression in the AKT2 gene. The high expression of AKT2 and the action of AKT2 protein kinase can prevent the cell apoptosis and increase the invasion of cancer cell. By now,the systemic researches on the relationship between the expression of AKT2 and histopathologic type, metastasis activity,angiogenesis of HCC have not been reported. And there are also no reports on gene therapy against angiogeneis of liver cancer. This study contains three parts of work: The first, the relationships between the expression of AKT2 in HCC and the tumor grade,tumor metastasis,angiogenesis of liver cancer. The second the inhibitory effect of AKT2 small interfere RNA on the growth of 7721 hepatocellular cell line (7721 cancer hepatocytes)in vivo. The third,the inhibitory effect of AKT2 small interfere RNA on 7721 cell in nude mouse.Part I:Study on the expression of AKT2 and its relationship to metastasis activity and angiogenesis of human HCC1 Expression of AKT2 gene in human HCC and normal liver tissue.The gene expression of AKT2 in 32 pair human HCCs and 4 normal liver were studied by RT-PCR method.The expressive rates of HCCs and normal tissue were 62.5%and 0% respectively.4 paired samples are yes or no.Moreover,The levels of AKT2 mRNA(AKT2 mRNA/GAPDH mRNA)were 0.59±0.01 in embolus, 0.62±0.03 in metastasis respectively.The difference between them are significant.The expression levels of AKT2 mRNA were positively correlated to pathologic grades,embolus and envelops of tumors.2 The protein expression of AKT2 in human HCC and normal liver tissue.The protein expression of AKT2 in 32 paired human HCCs and 4 normal liver were studied by immunohistochemistry.The expression rates of HCCs and normal tissue were62.5% and 0% respectively.The level of AKT2 protein was also significantly correlated to pathologic grades,embolus,capsules and HBsAg of the tumors.3 the expression of AKT2 in 4 cell lines were studied by Western-blot method.4 cell lines were all expressed,especially SMMC-7721.We find AKT2 are expressed in cytoplasm and cytomembrane mainly.It suggested that the AKT2 gene play an important role in the malignant progression of cancers.The overexpression of AKT2 and the correlation between AKT2 expression and tumor grade provided a useful parameter for evaluating the degree of malignancy at molecular level and for selecting the target gene in gene therapy.PartⅡstudy on designing siRNA of blocking AKT2 expression,construction expression vector and optimizing transfection conditionsIn studying siRNA of mammal cells,there are two key steps.One is to select effctive sequence of siRNA,the other is to transfer the sequences into cells effectively.We design the sequence in network.1 there is interaction between Liposome2000 quantity and plasmid quantity in transfection.There is highest efficiency when Lipofectamine2000 volume is 10ul and the palsmid of quantity is 4ug.The efficiency is over 85%. Transfection time is longer,cell cytotoxicity is bigger.The best time is 8 hours after transfection,the biggest transfection,the biggest transfection efficiency and the lest cell cytotoxicity.2 In the siRNA experiment we find the AKT2mRNA is lowest on the thirdth day by RT-PCR,but the AKT2 mRNA is not marked changed in the control teams.We also find the AKT2 protein is lowest on the fourth day by Western-blot but the AKT2 protein is not marked changed in the control teams.The plasmid, Supersilencing- AKT2–siRNA is more,AKT2 protein is lowest.We can draw the conclusion:the plasmid can be transferred into SMMC7721 cell efficiently and AKT2mRNA or AKT2 ptotein is induced.PartⅢThe inhibitory effect of AKT2-siRNA on the growth of HCC cells in vitroBecause there was no effective treatments on liver cancers,it is very urgent and important to find out new methods to solve this problem.According to the results in part I,we believed that AKT2 gene be a target in gene therapy of cancers.So we planed to utilize small interfere RNA technique to observe the inhibitory effects of small interfere RNA AKT2 mRNA on the expression of AKT2.1 We confirmed a/the overexpression of AKT2 in 7721 HCC cells by RT-PCR analysis and immunohistochemistry.We use 7721 HCC cells to Stable cell line which has plasmid Supersilencing-AKT2-siRNA or Supersilencing–AKT2-GFP growth rate among these groups had differences detected by MTT assay.2 The cell cycle is analysizd by FCM assay.The S stage of experimental team is longer than control team(P<0.05)but G0/G1 stage is shorter.The expression of CyclinD1,P27,P21 are reduced magnificently in experimental team by western-blot analysis.3 In adhesion experiment we find the adhesion capability of SMMC7721-siRNA cell is decreased 3 times or so.SMMC7721-siRNA cell invasion is decreased by Scribing test and Transwell test.blocking AKT2 also reduced the decreasing of expression of MMP-2,MMP-9.we find the expression of LNR and ICAM-Ⅰare inhibited marked in SMMC7721-siRNA cell line. but E-cadherin is increased markedly. We can draw the conclusion:blocking of AKT2 expression can inhibit growth and metastasis of SMMC7721-siRNA cell line.PartⅣstudy on blocking AKT2 protein expression to promote SMMMC7721 ApoptosisAKT2 is related to tumor Apoptosis closely.In order to study the value of AKT2 in gene theraphy,we observe the relationship between AKT2 and chemotherapeutics.Induction Apoptosis by CHX,we can see Apoptosis cell is increased in SMMC7721-siRNA cell line.By DAPI analysis,the Apoptosis rate is decreased markedly in in SMMC7721-siRNA cell line.(P<0.05)1 SMMC7721-AKT2-siRNA cell line processed with CHX for 24 hour,we find the apoptotic index (34.2%)is higher than control team(16.7%,20.4%).(P<0.05) by FCM.Outcome make it clear that blocking AKT2 can inhibit cell growth and enhance it sensitivity to chemotherapeutics.2 Processed with CHX,we find the protein Bax,Caspase3 of SMMC7721- AKT2-siRNA cell lines up-regulated(P<0.05),meanwhile Bcl-2 is downregulated by western-blot.3 Using equidensity chemotherapeutics Gem,we see growth inhibiting of SMMC7721-AKT2-siRNA cell line is higher than control team.(P<0.05). From above,we know blocking AKT2 can increase apoptositic sensitivity of hepatoma carcinoma cell to chemotherapeutics.This is foundation to the next in vivo experiment. PartⅤThe therapeutic effect of AKT2 small interfere RNA for HCC 7721 cells in vivoIn order to make further investigation on the role of AKT2 gene in the development of liver cancer and the effect of siRNA gene therapy in vivo,we carried out vivo study with BALB/nu/nu nude mice.15 nude mice were randomly divided into three groups and three prepared cells were subcutaneously injected on the back of mice:(1)control group:10 mice injected with wild-type 7721 HCC cells;(2) positive control group:5 mice injected with 7721 HCC cells transfected with plasmid GFP (3)experimental group:5 mice injected with 7721 HCC cells transfected with AKT2 small interfere RNA.The results showed:the transfected group were significently lower than that of positive control group and control group in volume,growth rate of tumors.In the tumors of experimental group, we knew that AKT2 protein were effectively blocked through analysis of wetern-blot.The present study demonstrated that AKT2 play an important role in the angiogenesis and development of HCC,clarified the therapeutic effect of AKT2 siRNA in vivo and suggested that AKT2 gene be a target for small interfere RNA gene therapy of liver cancer.We get a useful theory basis for gene therapy of liver cance...