Silencing effect study of Akt2-siRNA aimed at lung cancer cell NCI-H446.

Abstract

Lung cancer is one of the most frequent malignant tumors and at the first place of mortality in today's world, It becomes more and more clear that the initiation and development of lung cancer is multistep processes involved in many genes. RNAi gets more and more attention in gene therapy because of its high efficiency and specificity. With RNAi technology, eukaryotic expression vectors aimed at AKT2 siRNA was designed and constructed, which is transfected into human small lung cancer cell line NCI-H446. The silencing effect was observed. Using the known sequence of AKT2's mRNA (M95936), we designed two oligonucleotides that have short hairpin configuration and one oligonucleotide as control. DNA double strands was annealed, and cloned into vectorpGEM-T. Then, blue-white selection and T7/SP6 PCR was used to screen the positive clones, the target DNA and the express vector pRNAT-U6.2 were cut down by BamHI and XhoI restriction enzyme, and linked with each other; use PCR test to filtrate the positive clones and sequenced the target DNA, then transfected it into human small lung cancer cell line (NCI-H446), cultured and screened by G418, RT-PCR was used to detect the expression level of AKT2 mRNA in NCI-H446. validate the positive sequence by PCR and, transfect them to lung cancer cell line (NCI-H446), AKT2-mRNA's expression was evidently declined. The expression vector pRNAT-U6.2-AKT2 was constructed successfully and the expression of Akt2 mRNA in small lung cancer cell line (NCI-H446) was obviously silenced after transfection.