The Expression Of Human RNase-â…¢ Dicer In Primary Hepatocellular Carcinoma

The objective of this study is to observe the expression of the human RNase-ШDicer associated with RNA interference (RNAi) in primary hepatocellular carcinoma (PHC) and to find whether the expression of Dicer is connected with the continuous replication of hepatitis B virus (HBV) or with the malignancy of PHC.The subject is divided into three parts:The first part:To observe the expression of Dicer mRNA in PHC by real time fluorescent quantitative RT-PCR.Objective:To observe the expression of Dicer mRNA in PHC.Methods:To develop a specific,sensitive,reproducible,rapid,accurate and full-sealing real time fluorescent quantitative reverse transcriptase polymerase chain reaction (RT-PCR) method and to apply it to detect the expression of Dicer mRNA in human normal liver cell line WRL-68, hepatoma cell lines HepG2,HepG2.2.15 and 20 samples of PHC tissues and those adjacent ones. Results:Compared to human normal liver cell line WRL-68,the expression of Dicer mRNA in hepatoma cell lines HepG2,HepG2.2.15 is significantly reduced;Compared to the PHC adjacent tissues, the expression of Dicer mRNA in PHC tissues is significantly reduced too.Conclusions:The expression of Dicer mRNA in PHC is significantly reduced,regardless of cell lines or tissues.The second part:To observe the location and expression of Dicer protein in PHC. Objective:To observe the location and expression of Dicer protein in PHC. Methods:To detect the location of Dicer protein in human normal liver cell line WRL-68,hepatoma cell lines HepG2,HepG2.2.15 and 20 samples of PHC tissues and those adjacent ones by immunocytochemitry (ICC)and immunohistochemistry(IHC); To detect the expression of Dicer protein in human normal liver cell line WRL-68,human fetus liver cell line L-02,hepatoma cell lines HepG2,HepG2.2.15,Hep3B,SMMC-7721,Huh-7 and 20 samples of PHC tissues and those adjacent ones by semi-quantitative western blotting (WB).Results:Dicer protein is located at cytoplasm in PHC. Compared to human normal liver cell line WRL-68 or to human fetus liver cell line L-02,the expression of Dicer protein in hepatoma cell lines HepG2,HepG2.2.15,Hep3B,SMMC-7721,Huh-7 is significantly increased;and the expression of Dicer protein in PHC tissues of 20 samples is significantly increased too.Conclusions:The enzyme Dicer is located at cytoplasm in PHC. The expression of Dicer protein in PHC is significantly increased regardless of cell lines or tissues,nevertheless Dicer mRNA is significantly reduced.We guess there exists an amplification mechanism from transcription to translation.The third part:Through RNAi method,Dicer protein is reduced,we further to observe the relationship between the abnormal expression of Dicer protein and the continuous replication of HBV,or and the malignancy of PHC.Objective:To observe the relationship between the abnormal expression of Dicer protein and the continuous replication of HBV, or and the malignancy of PHC.Methods:HBV positive hepatoma cell line HepG2.2.15 is chosen as the experiment cell line, by means of RNAi method to target Dicer gene,then Dicer protein is observed and the indexes of the continuous replication of HBV,the malignancy of this cell line are determined,such as alpha fetoprotein(AFP), alanine aminotransferase(ALT), aspartate aminotransferase(AST) after RNAi. Results:The expression of Dicer protein in hepatoma cell lines HepG2.2.15 is significantly reduced after 48 hours' RNAi,at the same time there is no obvious difference in the indexes of the continuous replication of HBV,but the cell line reaches inhibition ratio to some extent and the indexes of the malignancy of this cell line except albnmin(ALB),AFP,ALT and AST are significantly reduced too.Conclusions:Generally speaking,Dicer protein is reduced after 48 hours' RNAi and at the same time there is no obvious change in the continuous replication of HBV, but the malignancy of HepG2.2.15 is reduced,so the high expressin of Dicer protein is not connected with the continuous replication of HBV but with the malignancy of this hepatoma cell line to some extent.