Research Of Aptamer U2 To Glioblastoma On Radiosensitivity And Inhibitory Effects Of U2-AuNP Complex To Glioblastoma In Vitro

Glioblastoma(GBM)is a malignant tumor with high malignancy and poor prognosis in brain tumors.The median survival time is only 14.6 months.The current treatments mainly includes surgical resection combined with preoperative and postoperative chemotherapy and radiotherapy to improve the survival of patients.Because glioblastoma has poor borders as well as high invasive ability and the high recurrence rate after resection,combined with radiotherapy and chemotherapy resistance all occur during the treatment to GBM,which make the therapy of glioblastoma more difficult.It is time to find effective targeted therapy in the field of glioma research.Amplification and mutation of the epidermal growth factor receptor are common in glioblastoma.Epidermal growth factor receptor(EGFR)and its mutant EGFRv Ⅲ amplification and overexpression increase the tumorigenicity of GBM;through autophosphorylation,EGFRvⅢ activates its downstream signaling pathway without binding to the ligand epidermal growth factor,which promote glioblastoma proliferation and invasion.Aptamers,which are single-stranded oligonucleotides that have been screened by SELEX technology,are attracting more and more attention by researchers due to their high affinity and high specificity to their targets.Due to the excellent optoelectronic properties,gold nanoparticles have been widely studied in drug delivery systems.The main purposes of our study are to explore the mechanism of radiosensitivity of aptamer U2 to glioblastoma and the anti-tumor effect of aptamer U2 in conbination with gold nanoparticles to glioblastoma innitially in vitro.In this study,we first verified the specificity of aptamer U2 to U87MG target cells over-expressing EGFRvⅢ(U87-EGFRvⅢ cells)by flow cytometry,which was screened by cell-SELEX technique in our previous work,and explored the mechanism of aptamer U2 entry into U87-EGFRvⅢ cells by cell immuno-staining.The molecular mechanism of aptamer on the inhibition effects of proliferation,migration and invasion to U87-EGFRvⅢ cells was studied by western blot.Through the combination of X-ray,we want to figure out the role of aptamer U2 play a role as the radiosensitizer.To detect the radiosensitivity effects of aptamer U2,we underwent clone formation assay and comet assay,and explored the molecular mechanism of radiosensitization by western blot.Finally,we conjugated the aptamer and gold nanoparticles by Ultraviolet-visible(UV-VIS)spectrophotometer,transmission electron microscope(TEM),dynamic light Scatterometer(DLS)and agarose gel electrophoresis to detect whether the aptames successfully conjugate with the gold nanoparticles and then explored the anti-tumor effects in vitro.The binding rate of FAM-U2 to FAM-Scrl was found to be less than 5%by incubating various glioma cell lines with FAM-labeled aptamers and counting by flow cytometry.FAM-U2 and U87-EGFRvⅢ cell binding rate as high as 95%;through the confocal laser scanning confocal microscope,it had been proved that U2 can be uptaked into the U87-EGFRvⅢcells and the complex of U2-EGFRvIII-endosome co-localization is found intracellular.The results of western blot showed that U87-EGFRvⅢ cells could significantly reduce the phosphorylation level of EGFRvIII protein without changing its total protein level at the same time after the U2 treatment,while no high expression of PDGFRβ was detected.The level of phosphorylation of Met and ERK protein downstream of the EGFRvIII protein detected after U2 treatment is also significantly reduced.In the study of radiosensitization,aptamer U2 combined with X-ray significantly reduce the clonogenic rate of U87-EGFRvIII cells and increase the production of DNA fragments;as shown in comet assay,the comet tail dragged obviously,tail DNA content,tail moment and Olive moment increased.By western blot we found that U2 combined with X-ray inhibits DNA damage repair signaling,the phosphorylation level of pH2A.X,ATM,Chk2 and 53BP1 total protein levels.At the same time,the level of EGFRvⅢ protein was detected and the level of phosphorylation was also significantly decreased after U2 treatment.In the study of aptamer U2 in conjugation with gold nanoparticles,it was observed that the stability both of gold nanocrystals and aptamer in solution were improved after the modification.The results of TEM,DLS,UV-VIS spectrophotometer and horizontal agarose gel electrophoresis confirmed that the gold nanoparticle was successfully connected to the aptamer,and the maximum absorption peak of gold nanoparticle was red-shifted.The proliferation ability of U87-EGFRvⅢ cells was detected by EdU staining and we found that the U2-gold nanoparticle(U2-AuNP)complexes significantly inhibited the proliferation ability of the target cells.All in all,the data above demonstrated that the aptamer U2 inhibits the proliferation,migration,invasion and apoptosis of U87-EGFRvⅢ cells by decreasing the phosphorylation level of EGFRvⅢ protein and further inhibiting the downstream signaling pathway.Aptamer U2 enhances the radiosensitivity to gliomas which may be used as a radiosensitizer in clinical practice;the preparation of gold nano-aptamer complexes inhibit the growth of U87-EGFRvⅢ cells much more efficiently,which is beneficial to explore the anti-tumor effects of U2-AuNP in vivo.