Construction Of RNA Interference Plasmid Of Ilk Gene And Its Effection On Biological Activity Of Lung Cancer A549Cell Line

Objective: To construct the expression vectors of siRNA which canspecifically inhibit the expression of ILK gene and select out the mosteffective one. Then detect its effection on the biological activity of A549cellline.Methods: Based on the gene sequence of ILK, three pairs of siRNAoligonucleotides were designed, then cloned into pGenesil-1vector toconstruct pGenesil-ILK1, pGenesil-ILK2and pGenesil-ILK3recombinantplasmids respectively, which were stably transfected into A549cells afterconfirmed by restriction enzyme digestion and sequencing. The expressionof ILK mRNA and protein in each group was detected by RT-PCR andWestern-blot, then the RNA interference efficiency was analyzed. Takingthe most effective one as experimental group, the cell proliferation andapoptosis was detected by Flow cytometry and MTT assay respectively.Results: The result of restriction enzyme digestion and sequencingproved that pGenesil-ILK1, pGenesil-ILK2and pGenesil-ILK3recombinant plasmids were constructed successfully. After these plasmidsstably transfected into A549cells, the expression of ILK mRNA and protein decreased significantly, and the pGenesil-ILK1group was the mosteffective one. The results of Flow cytometry and MTT assay suggested thatthe apoptosis activity of A549cells in experimental group increased and itsproliferation activity decreased significantly (p<0.05).Conclusion: The expression vectors of siRNA which can inhibit theexpression of ILK gene of lung cancer A549cells efficiently andspecifically were constructed successfully. The inhibition of ILK geneexpression of A549cell can promote its apoptosis and inhibit itsproliferation significantly, which lay the foundation for lung cancertargeted gene therapy research.