ShRNA Inhibition Of Human Endometrial Cancer Cell Line Ishikawa Cells Her-2/neu Gene Expression

ObjectiveObservation of vector-mediated shRNA interference technology can effectively inhibit human endometrial cancer cell line Ishikawa HER-2 gene expression.RT-PCR Detection of experimental and control groups between the mRNA level after transfection whether there is any difference between.Western Blot Detection of experimental and control groups between the P185 transfected whether there are differences in protein expression.Materials and MethodsIshikawa cells purchased from Cell Institute at the Xiangya School of Medicine. Ambion provided the use of RNAi design tool designed for the HER-2/neu gene in two specific short hairpin RNA(shRNA),GC ratio of 45%to 55%between.Selection of pSilencer 4.1-CMV vectors(Ambion) as a plasmid vector to construct the two specific for the HER2 gene in the recombinant plasmid.First generation of cells,spread to a sufficient number of cells frozen back-up.The use of E.coli strain JM109 as the state felt a great deal of plasmid amplification in order to reserve a sufficient number of plasmids,the transfection media lp2000 as Ishikawa cells.Test is divided into three groups,namely,shRNA-HER2-1 group,shRNA-HER2-2 group and the expression of non-specific short hairpin shRNA-CON eukaryotic expression vector of the negative control group.Three sets of shRNA eukaryotic expression vector transfected human Ishikawa endometrial cancer cells,RT-PCR assay after transfection 24,48,72 h HER2 gene mRNA.Western Blot Detection of transfected gene 24,48,72 h HER2 protein expression of P185,compared to the differences between the three groups.ResultsConfirmed by the identification of eukaryotic expression vector of shRNA-HER2-1,shRNA-HER2-2 and shRNA-CON were successfully constructed. RT-PCR results showed that after transfection 24h,48h and 72h after the Her-2/neu 1 and Her-2/neu 2 group decreased mRNA levels of specific non-Her-2/neu con group 70.1%and 80.2%,60.2%and 71.2%and 69.3%and 78.5%,confirmed by the two recombinant plasmid-specific mRNA levels in the her-2 can inhibit gene expression. And Western Blot Detection of P185 protein level decreased non-specific Her-2/neu con group 79.1%and 82.3%,57.2%and 67.8%and 68.4%and 73.3%,also confirmed that the two chain-specific protein levels in the inhibition.Compared to a more pronounced inhibition of the chain.Conclusions1.ShRNA-HER2 and the two negative control shRNA-CON eukaryotic expression vector was successfully constructed;2.ShRNA-HER2-1,shRNA-HER2-2 can be found in the mRNA effectively inhibited Ishikawa cells,the level of HER2 gene expression.3.ShRNA-HER2-1,shRNA-HER2-2 can effectively inhibit the expression of P185 protein.4.RNA interference technology for gene therapy of endometrial cancer provides a new strategy.