Silencing Effect Study Of Akt2-siRNA Aim At Lung Cancer Cell NCI-H446

Background and aims:Lung cancer is one of the most frequent malignant tumors and at the first place of mortality in today's world. The collective survival percentage of lung cancer sufferer was not ameliorated evidently in spite of the technology developed rapidly in the past 10 years. Operation, chems and radiotherapy have mainly been adopted to cure lung cancer now, biology targeted therapy is the breakthrough and researchful hot spot of lung cancer's therapy, Molecular targeted drug mainly aimed at nonsmall cell lung cancer. The scientific operators are cognizanted more and more clearly that the happeneese and development of lung cancer is a participant course of multigene and multistep, as one of the new high efficiency, strong difference therapeutic instrumentality, RNAi get more and more recognitions in gene therapy.RNA interference (RNAi) is the phenomena of sequence-specific posttranscriptional gene silencing triggered by double stranded RNA (dsRNA) with 21-23nt nucleotide acid in longth. It' s effected mechanism could de generalized about that: introduced or endogenous dsRNA is incised to double small interference RNA(siRNA) with 21-23nt nucleotide acid by Dicer enzyme, a dsRNA-specific endonuclease. Double siRNA combine with one proteinic complex to form into the RNA-induced silencing complexes (RISC), Under the action of helicase RISC was actived by ATP offer energy, siRNA untie it's double strand, the sense strand is released, the antisense strand lead the actived RISC identify and combine specifically with target mRNA, it incise mRNA, exonuclease decompose the incised segment thoroughly so that restrain the expression of mRNA.Akt is one of gene coding Ser/THR protein kinase. It is named protein kinase B(PKB) because it's proteinic production has most approximate with PKA and PKC in catalysed domain.Under the adjustment of numerous upriver regulatory facters, Most of it's numerous underriver substrates is proved to oncogene,growth factor,proapoptotic factor,angiogenesis facter etc now. So Akt take more important action on occurrence,transfer,drug-resistance,radioresistance of tumor. Akt2 have upper incidences in the pancreas cancer,breast cancer,ovary cancer and lung cancer. So we target Akt2 gene, silence this gene from the technology of siRNA, and offer laboratorial elements for genic function and therapy of lung cancer.Methods:Using the known genic sequence of Akt2's mRNA (M95936), from filtration of BLAST, design and compose a siRNA that have short hairpin configuration, connect this constructed genic segment with clone vector-pGEM-T Easy, Then, blue-white selection test and T7/SP6 PCR were used to screen the positive clones(pGEM-T-Akt2), then the target DNA was sequenced. After this, 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(pRNAT-U6.2-Akt2),then the siRNA express vector pRNAT-U6.2-Akt2 was transfected into human small lung cancer cell line(NCI-H446), cultured and screened by G418,transfered effect was observed by fluorescent microscope, RT-PCR was used to detected the express level of Akt2 mRNA in NCI-H446 .Result:1.Fore screen of BLAST, 19 bases oligonucleotides which isGGTGTCTGTCATCAAAGAA (96-114nt) were ascertain from useing the knowngenic sequence of Akt2's mRNA (M95936) of GenBank.2.After the hairpin DNA of siRNA annealing, the results of agar-gel electrophoresisanalysis showed that there was an obvious stripe whose molecular weight wassimilar to the expected.3.The annealing products was recombined with pGEM-T Easy, which wastransformed into JM109. After screened and identified, the positive clone(pGEM-T-Akt2) was gotten.4.After sequenced, the DNA sequences inserted the recombination pGEM-T-Akt2was conformed completely with the design. 5.After sub-clone, the target DNA and the express vector pRNAT-U6.2 were cut down by BamHI and XhoI restriction enzyme, and linked with each other; the positive clone was gotten form PCR with the primer desigened by the company.6. The Akt2 mRNA level in NCI-H446 was significant different among transfected, pRNAT-U6.2 and untransfected. The expression in experimental group which transfected pRNAT-U6.2- Akt2 was nearly inhibited. Express level of others was still high.Conclusion:1. The siRNA oligonucleotide possessed of hairp and targeted Akt2 gene was designed and composed successfully.2. The express vector pRNAT-U6.2- Akt2 was constructed successfully3. The express of Akt2 mRNA in small lung cancer cell line(NCI-H446) was obviously silenced after pRNAT-U6.2- Akt2 transfected.