Hepatoma Cells, Rhoa Small Interfering Rna Vector Construction And Identification

The incidence rate of the primary liver cancer is high,the surgery excision is the main method of treatment,but after the operation the recurrence rate is so high that it has become the most crucial prognostic factor for the patients.The invasion and metastasis of liver cancer is one of the main influence factors,further study of the invasion and metastasis of liver cancer will play a very important role in the liver cancer patient's overall prognosis.The Rho family who belongs to the Ras ultra family is an important intracellular signaling molecule.It plays a critical role in cellular fundamental vital activity.The Rho protein has the close relationship with the occurrence of the tumor.It participates in the tumor's infiltration and mestastasis.The present studies confirm that the RhoA's expression is exceptionally high at the colon cancer,breast cancer,the lung cancer,the head and neck cancer, liver cancer and so on.This research interfers the RhoA protein's expression and the way of the signal transduction and analyses the tumor cell's multiplication and the cell cycle's change using the RNAi technology.It provides the experimental model for further studies of the stress textile fiber's changement,the tumor cell's distortion,the migration,the adherency and the attack ability's changement.Finally they can understand the relationship between RhoA and liver cancer's invasion and metastasis.This study includes two parts:Part 1 Construction of recombinant plasmid with hoA-targeted short hairpin RNAObjective:To construct the recombinant plasmid expressing RhoA -targeted short hairpin RNA (shRNA)by pGenesil-1 plasmids vector.Methods:Two pairs of DNA sequences were designed,and then synthesized into complementary chains by annealing,respectively.Then the obtained products containing short hairpin structure were inserted into plasmid vector pGenesil-1 with U6 promoter.The recombinant plasmids were transformed into Escherichia coli strain DH5a for screening and amplifying.The sequence analysis of the plasmids identified by restriction enzyme was carried out.Results:The two RhoA-targeted shRNAs were successfully inserted into the plasmid vector pGenesil-1,and the coding sequences of the obtained shRNA were consistent with the designed fragments.Conclusion:The recombinant plasmid of RhoA-targeted shRNA is successfully constructedPart2 construction and identification of RhoA specific siRNA expression vector in hepatocellular carcinoma cellObjective:To construct RhoA siRNA plasmid expression vector.Methods:Cultures human high-metastatic hepatocellular carcinoma cell line HEPG2,accoding to the computer aided design,RhoA specific siRNA gene was synthesized and cloned into the RNAi-Ready Pgenesil-1 Vector.The constructed RhoA-RNAi plasmid were transfected into human HEPG2 cell,Western blot detected the effected of RhoA-RNAi plasmid.Result:The recombinant was cloned and the sequence was obtained.RhoA- RNAi plasmid can down regulated the expression of RhoA in human high-metastatic hepatocellular carcinoma cell line HEPG2.Couelution:Susccessfully cloning the recombinant make it possible searching new mechanism of RhoA in hepatocellular carcinoma.