The Effects Of A DNA Aptamer To U87-EGFRv? Cells On The Proliferation,Apoptosis,Migration And Invasion Of Glioma

Glioma is one of the most common malignant intracranial tumor in the brain.The tumor cells infiltratively grow in vivo with high degree of malignancy,complex clinical manifestations and poor prognosis.The effect of traditional treatments including surgery,radiotherapy,chemotherapy and a combination of radiotherapy and chemotherapy are extremely limited.Glioma is still difficult to be cured.The median survival time of patients is not longer than one year and a half.Early diagnosis and prognosis both are helpless.The primary reasons for gliomas causing the death of patients are the ability of abnormal activation and migration invasion of tumor cell.Thus,finding a drug that is capable of inhibiting glioma cell growth,proliferation,migration and invasion as an effective treatment is quite significant.With the rapid development of molecular biology,molecular targeted therapy has been more and more popular in clinical treatment of malignant tumor,and become a new method of treatment.Molecular targeted drugs can select the high specificity oncogenic sites,and bind target with high affinity in vivo,in order to destroying its structure and function or directly killing tumor cells without damaging other cells of surrounding normal tissues.Moreover these drugs have good stability and less toxicity.The common manifestation in glioma patients is increased expression and mutation of epidermal growth factor receptor(EGFR).The protein that EGFR encoded is a type I receptor tyrosine kinase(RTKs).It plays an important role in membrane information transfaction.EGFR is over expressed in a variety of malignancies,and related to the degree of malignancy.However,EGFR expression is also found in normal tissues,limiting the EGFR-targeting therapy.There are three kinds of EGFR mutations,EGFRvI,EGFRvII,and EGFRvIII.The most common mutation is EGFRvIII.EGFRvIII is shorter than EGFR,so that it is not regulated by ligands to produce EGF(epidermal growth factor)signals and be activated sustainable.Clinical studies have demonstrated a correlation between EGFRvIII expression and poor prognosis,chemoresistance for patiens.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.Also they are specificly expressed in cancer cells but not in normal cells therefore it is a good target for cancer therapy.Aptamers are single-stranded ligonucleotides which are selected from large-capacity random ssDNA or RNA library by systematic evolution of ligands by exponential enrichment(SELEX).Screening aptamers are capable of binding to specific target molecules with high affinity and specificity.They have many attractive features as molecular probes compared with that of conventional antibodies:(1)low molecular weight,long-term stability;(2)low immunogenicity and toxicity;(3)high affinity and specificity;(4)quick and reproducible synthesis and modification.Aptamers can be used effectively on targeted therapy,detection and diagnosis of glioma.These advantages have made aptamer an excellent alternative as molecular probe for clinical applications.The malignant glioma cells U87MG,U87-EGFRwt cells over expressing EGFR and U87-EGFRv? cells over expressing EGFRv? were cultured.Before this study we used the mothed of streptavidin-biotin magnetic beads separation to separate single-stranded DNA library and finally obtain a aptamer U2 which has high purity affinity and specificity(Kd=6.27±1.40nM).To explore aptamer U2 binding ability of U87-EGFRv? cells and the effects of inhibitions of U87-EGFRv? cells,we started the following experiment research.Research is divided into three parts,the first part is observing and detecting the binding of aptamer U2 and U87-EGFRv? cells by flow cytometry and confocal microscopy;the second part is to explore whether aptamer U2 is able to affect the growth,proliferation,apoptosis,migration,invasion and other physiological characteristics of U87-EGFRv? cells;the third part is to explore the mechanism of aptamer U2.Firstly,we investigated whether U2 binds to EGFRv?.We synthetized the 5'end FAM-labelled aptamers U2,FAM-labelled original library GN was used as control.U87-EGFRv?,U87MG and U87-EGFRwt cells were treated with 1?M FAM-U2 or GN for 24h.Accordingly to the findings,FAM-U2 bound to U87-EGFRv?,by flow cytometry at a higher extent(relative combined rate 94.0333%±0.1764)(P<0.001)than FAM-GN bound to U87-EGFRv?(relative combined rate 7.4%±3.4646).Further,it recognized U87-EGFRvIII cells but not U87-EGFRwt cells and U87MG cells thus confirming its specificity for U87-EGFRv? cells.Next,we analyzed the binding of the fluorescent FAM-labelled U2 to EGFRvIII on the surface of U87-EGFRvIII cellsby confocal microscopy.Aptamer U2 localizes at membrane level of EGFRv?,showing puncta of colocalization with EGFRv? after only 5 minutes incubation whereas multiple U2 dots were accumulated in the cytoplasmic side of cell membrane in 20 minutes incubation.Aptamer binding seems to be highly specific for EGFRvIII and through endocytosis mechanism possibly.We analyzed the morphological change of U87-EGFRv? cell after transfection concentration of 50 nM aptamer U2,in order to explore if the aptamer U2 have any influence on the physiological characteristics of U87-EGFRvIII cell.U87-EGFRvIII cells that be treated with aptamer U2 have occurred typical cell apoptosis including wider intercellular space,the condensation cells and the apoptosome.To evaluate the biological significance of the U2-dependent EGFRv? inhibition,we investigated whether the aptamer could affect cell growth,migration,invasion and apoptosis of U87-EGFRvIII cells overexpressing EGFRvIII.We performed CCK8 experiments to determine whether the long-term U2 treatment alters the cell viability of EGFRvIII-expressing GBM cells.Fold change viability=(treatment group OD-blank group OD)/(control group OD-blank group)× 100%.Significant decrease(P<0.001)in cell viability was observed in U87-EGFRv? cells treated 24h with U2 concentration of 25,50nmol/l(Fold change viability 59.9360%±12.2423,51.8643%±3.3743).We found that the aptamer U2 reduced the survival of U87-EGFRvIII cells but not U87MG cells and U87EGFRwt cells.Aptamer caused a time and dose-dependent inhibition of U87-EGFRv? cell viability.We then performed Annexin V-FITC/PI experiments to determine whether the U2 treatment is able to lead to apoptosis of U87-EGFRv? cells.We observed that aptamer raised the apoptosis rate of U87-EGFRv? cells significantly(apoptosis rate 50.55%±1.5804)but not U87MG cells and U87-EGFRwt cells cells.To evaluate whether the aptamer U2 could affect migration and invasion of U87-EGFRv? cells,first,by using a scratch-wound assay that measures cell motility.We observed thatU87-EGFRv? cells treated by U2 still had a wide gap at 8 hours after scratch(wound closurel 1.548%±1.6400).The wound of U87MG cells treated by U2 was barely visible(wound closure21.171±3.5290).These data has statistical significance(P=0.048).At 24 hours after scratch,the wound of U87MG cells almost closed(wound closure21.2%±1.7760)compared with U87-EGFRv?cells treated by U2(wound closure71.515%±1.8280).The U87-EGFRv? cells treated with U2 significantly delayed the wound closure compared to the control cells treated with U2.Further,migration was analyzed by a "transwell migration assay" that assesses the chemotactic capacity of cells.The benchmark is the number of cells,treated by GN,going through transwell membrane.After treated 24h,as compared to U87MG cells treated with U2(precentage of migrated cells 93.917%±3.4010),Precentage of migrated U87-EGFRvIII cells was reduced to 41.94%±3.0000(50nM).And precentage of migrated cells 34%±3.0000 of U87-EGFRv? cells and 96.946%±6.4100 of U87MG cells at 48 hours,respectively(P<0.001).Accordingly to the above findings,aptamer U2 is capable to reduce the migration ability of U87-EGFRv? cells.We then examined the effect of U2 on the capacity of the cells to invade through a matrigel-coated membrane by a "transwell invasion assay",which has been reported to mimic the whole process of cell invasion of basement membranes.Using this assay,we observed that the invasion rate of U87-EGFRv? cells in the presence of U2 treatment 24h was significantly decreased(precentage of invaded cells 38.9222%±2.2670)compared with U87MG cells(precentage of invaded cells 103.3079±12.8028).36.1027%±0.5160 of U87-EGFRv? cells and 96.206%?4.0890 of U87MG cells at 48 hours,respectively(P<0.001).These results emphasize the use of the aptamers U2 to affect U87-EGFRvIII cell motility,migration and invasion.At last,we detected whether U2 interferes with the autophosphorylation activity of EGFRvIII in U87-EGFRvIII cells to attenuated EGFRvIII activation and downstream signaling.To minimize EGFR basal(unstimulated)phosphorylation,cells were maintained in low(2%)serum concentration for 6 hours before aptamer treatment.In these conditions,EGFRvIII was constitutively phosphorylated while basal tyrosine phosphorylation of EGFRwt was undetectable.U2 treatment(200 nM)of U8-EGFRvIII cells attenuated activation of phosphorylation of EGFRvIII following 6 hours-incubation,where it did not affect the total level of the EGFRvIII protein.Total and phospho-EGFRvIII levels were unaffected by GN negative control.A substantial reduction of phosphorylation of EGFRv? downstream effectors extracellular signal-regulated kinase(ERK)was observed upon U2 treatment of the cells suggesting a critical role of EGFRv? signaling.Altogether,these results show the ability of U2 aptamer targeting EGFRvIII to affect U87-EGFRv? cell biological significance including cell viability,migration,invasion and apoptosis.The activation and downstream signaling is still to be investigated.This study provides research foundation for the development of molecular targeted therapy,and offers new way and ideas for the treatment of glioblastoma.