Selection Of U87-EGFRvⅢ Cell Specific Aptamers Which Mediate SiRNA Delivery

Glioblastoma multiforme (GBM) is one of the most common and malignant brain tumors. The prognosis for patients with GBM is very poor:adults diagnosed with this condition have1-,3-and5-years relative survival rates of less than30%,5%and3%respectively. Epidermal growth factor receptor variant Ⅲ (EGFRvⅢ) is a naturally occurring mutant of EGFR and is expressed on approximately20%to30%of GBMs. Clinical studies and in vitro studies also have demonstrated a correlation between EGFRvIII expression and poor prognosis, chemoresistance for patients. EGFR and EGFRvIII which enhance tumorigenic behavior and increase malignancy are also commonly found in many tumors such as non-small cell lung cancer (NSCLC), colorectal cancer and colon cancer. So they can be treated as main targets for oncotherapy. Monoclonal antibodies were used in clinical treatment as they can block EGFR pathway. As tyrosine kinase activity inhibitors, monoclonal antibodies have given promising results in preclinical and clinical trials. However side effects of these therapies occur as EGFR is aslo expressed in normal tissues. Moreover, tyrosine kinase activity inhibitors, which are of the immunogenicity as antibodies, also produce side effects. It is important to develop new phamaceutical preparations which are lack of immunogenicity, low molecular weight, less toxicity, good targetability and good stability. It is reported that EGFRvⅢ is not expressed in normal cell, and may be regarded as an effective target for the immunotherapies. Aptamers targeting EGFRvIII may provide a good solution because of the high specificity and lack of immunogenicity.Aptamers are single-stranded oligonucleotides capable of binding to target molecules with high affinity and specificity due to their tertiary structures. They have many attractive features as molecular probes compared with that of conventional antibodies:low molecular weight, quick and reproducible synthesis, easy modification, long-term stability, low toxicity, low immunogenicity, and fast tissue penetration. These advantages have made aptamer an excellent alternative as molecular probe for clinical applications. Aptamers are selected form random ssDNA or RNA libraries with approximately1013-1015diversity through cycles of in vitro affinity selection and amplification known as systematic evolution of ligands by exponential enrichment (SELEX).Recent studies have reported that some aptamers are screened against complex targets, such as cell membrane proteins or whole cells (cell-SELEX). When it comes to being molecular probes for cancers, cell-SELEX has many advantages over protein-SELEX. There is no need to purify and immobilize molecular targets and the targets can maintain native folding and glycosylation sites at the surface of cells. Moreover, new molecular probes can be obtained even without prior knowledge of potential target molecules of cancer cells.Aptamers with high specificity and affinity against U87-EGFRvIII cell line overexpressing EGFRvIII were selected from synthesized30nt random oligonucleotide library through cell-SELEX. Meanwhile we resort to establish a cell based enzyme-linked assay (cell-ELA) method for detecting the affinity of the U87-EGFRvIII cell specific aptamers. By using confocal microscope, aptamers were observed to be endocytosed by the membrane receptors of the U87-EGFRvIII cells. Therefore, we postulated that aptamers can be regarded as an effective vehicle for the delivery of c-Met siRNA which can mediate post-transcriptional gene silencing (PTGS).Firstly, U87-EGFRvIII cells overexpressing EGFRvIII was cultured. In vitro, we constructed an oligonucleotide library as an initial library which has a length of76nt base, which comprises fixed sequence at both ends of oligonucleotide and30nt of random sequence in the middle. Cultured U87-EGFRvIII cells was as an target and incubated with the initial library. After being washed, the ssDNA binding with the U87-EGFRvIII cells was isolated and amplified by asymmetrical PCR. The purified PCR products were the ssDNA library for the next round. Aptamers with high specificity and affinity to U87-EGFRvIII cells obtained by increasing the intensity of screening (increasing the salmon DNA amount, shortening the time of incubation and increasing the frequency of vibration, etc.). From the fourth round, the U87cells were used as negative cells. The ssDNA library which did not combine with the U87cells was as a new screening library incubated with the U87-EGFRvIII cells. After14screening rounds, aptamers binding U87-EGFRvⅢ cells specifically were obtained.The affinity of ssDNA library labeled with biotin was determined by cell-ELA. The last screening library was amplified, purified, cloned by T vector and sequenced. The related softwares such as RNA STRUCTURE5.2were employed to analyze the primary structure and predict secondary structure of the aptamers.In order to determine the affinity of the aptamers, FITC was labeled, the fluorescence was measured after aptamers were incubated with U87-EGFRvIII cells. And the fluorescence intensity was also determined by flow cytometry assays. Then we determined the affinity of aptamers by using a cell-ELA method. The Kd value was calculated by Sigmaplot12.0software.Imaging of cell-aptamer complexes was observed by confocal microscope with a40x objective. Aptamers which could be endocytosed by U87-EGFRvIII cells were found. Subsequently, the molecular targets of these aptamers were found by pull down experiment. Biotinylated aptamers were stabled by the streptavidin magnetic beeds, and then incubated with U87-EGFRvⅢ cell lysate. At last, the streptavidin-biotin compound was heated and then isolated by SDS-PAGE gel. The proteins which interacted with aptamers were transferred to PVDF membrane. Then the membrane was incubated with EGFR antibody and developed by ECL. If aptamers can bind the EGFRvⅢ,145KDa band would be displayed. We hope these aptamers can mediate the delivery of c-Met siRNA. Lipofectamine2000served as positive control and the c-Met protein was measured by western blot experiment.The results:steady increases in optical density on U87-EGFRvIII cells were observed with increasing selection number. There is a significant difference compared the12th,14th with the first round(p<0.05). Once the binding sites between U87-EGFRvIII cells and ssDNA enrichment library saturated, the binding rates would not increase. After cloning and sequencing, the structures of the ssDNA were analyzed, and the analysis of the secondary structure indicated all sequences mainly have stem-loop or hairpin structure. We found that10sequences appeared twice. We chose5sequences (A41, A43, A32, A47, A15) randomly among the10sequences which were labeled by FITC or biotin for further study.The FITC labeled aptamers separately incubated with U87-EGFRvIII, U87, HEK293, and then aptamers specifically binding to the U87-EGFRvIII cells were observed by fluorescence microscope. We found that the fluorescence of A41and A32appeared on the nuclei, while the others appeared around the menbrane. Transportation to the perinuclear region could have occurred through endocytosis. Then the equilibrium dissociation constants (Kd) was determined by Cell-ELA. The kd value of the A43, A41, A32, A47, A15were4.40±0.27nM,37.57±6.01nM,0.62±0.04nM,3.33±0.39nM,14.09±2.95nM respectively, and they were in the range of pmol/L to nmol/L. A32has the same high affinity compared with the EGFR antibody (Kd=0.32±0.07nM). According to the results of the pull-down and western blot experiments, we found A32bind with the EGFRvⅢ.Many researchers have reported that one of the most interesting tumor markers is the tyrosine kinase receptor c-Met, a transmembrane receptor protein whose primary stimulatory ligand is hepatocyte growth factor (HGF). HGF/c-Met are not only involved in the development and morphogenesis of embryonic tissues, but also affect tumorigenicity, malignant progression and tumor angiogenesis by inducing cell cyle progression, cell adhesion, cell migration, invasion, and antiapoptotic responses. HGF/c-Met are associated with the development and progression of GBM, and overexpression or misexpression of c-Met often correlates with a poor prognosis. It was reported that c-Met overexpression was found in significantly more recurrent GBM(15/19) than primary GBM(7/19, p=0.020). Recurrent GBM specimens had obviously higher expression levels of c-Met.A32specifically bind with the EGFRvⅢ on the U87-EGFRvⅢ cells and has high affinity. Maybe A32can be endocytosed by EGFRvIII. The biotinylated A32can be connected with c-Met siRNA by a bridge of streptavidin. C-Met siRNA transfected by lipofectamine2000was served as a positive control, the c-Met siRNA was delivered to the U87-EGFRvⅢ cells by A32. Positive control group(lipofectamine2000):The concentration of siRNA is100nM. There is significant decrease of the expression of c-Met after48h transfection whether streptavidin was added or not(0.631±0.08, p=0.051;0.524±0.04, p=0.08). Experimental group(A32):Compared with the control group, there was no significant declining expression of c-Met after48h (40nM and80nM of siRNA). When the concentration of siRNA is100nM, the significant decline of c-Met protein expression was observed by western blot after72h (0.57±0.09, p=0.039). The experiments demonstrated that the A32can deliver the c-Met siRNA to the U87-EGFRvIII as a vehicle and result in the post-transcriptional gene silencing. The method that aptamers connect with siRNA by streptavidin was effective. No cytotoxicity of streptavidin was observed.In this study, we demonstrated that DNA aptamers can be used as an efficient and targeted nucleic acid carrier for the siRNA delivery. This is a new method for siRNA therapy. Meanwhile, it is important for the diagnosis and treatment of GBM with the use of anti-U87-EGFRvIII aptamers. All of these results will lay the foundation for finding new method for target therapy.