Construction Of Anti-PSMA Aptamer Mediated Autophagy And Pro-apoptotic System And Its Preliminary Identification Of Anti-prostate Cancer Effects

Objective: To construct recombinant plasmid consisting of streptavidin?SA? and evolutionary conserved district?ECD? of atutophagy-related gene Beclin1. And to express and purify SA-ECD fusion protein. Then, anti-PSMA aptamer mediated autophagy and proapoptosis system was conjugated with NuBCP-9-r8, and the preliminary function on prostate cancer cells was validated.Methods: Codon optimized fusion gene SA-ECD with initiation codon ATG and a His-tag in front of SA-ECD, in total 1011 bp was desiged, synthesized and cloned into vector p UC57. Then it was inserted into the prokaryotic expression vector p ET30 a with Nde I and Hind? by polymerase chain reaction?PCR? and positive clones were identified by double restriction enzyme digestion and DNA sequencing.Correct sequence of the p ET30a-SA-ECD was transformed into BL21 as expression strain, choosing the final concentration of IPTG 1mmol/L, 15? inducing 16 h, 37? inducing 4h as expressive conditions. Fusion protein SA-ECD purified by Ni-NTA agarose and detected by Western-blotting.Cell-penetrating experiments identified eight polyarginine?R₈? in penetrating efficiency;Using the efficient and specific binding properties between streptavidin-biotin to construct anti-PSMA aptamers targeting autophagic and pro-apoptotic system, which assembled by SA-ECD fusion protein,biotin labeled anti-PSMA aptamers and NuBCP-9-r8 peptides. And electrophoretic mobility shift assay?EMSA? was used to detect the binding between SA-ECD and biotin labeled anti-PSMA aptamers/NuBCP-9-r8 peptides; CCK-8?cell counting kit-8?, flow cytometry?FCM?, Trypan blue assay, morphological analysis, Acridine orange staining and Western blotting to validate its preliminary function of autophagy and apoptosis in prostate cancer cells.Results:Double restriction enzyme digestion and sequencing results showed that the recombinant plasmid p ET30a-SA-ECD was successfully constructed; The His-tagged fusion protein induced by IPTG was efficiently expressed in E.coli, lower temperature and longer time were beneficial to protein expression. So the final concentration of IPTG 1mmol/L, 15? inducing 16 h was chosen as optimized conditions to expand protein expression; SDS-PAGE analysis of the fusion protein expressed mainly as inclusion bodies, solubility less than 10%; for this reason,we zoom expression system, culture supernatant SA-ECD fusion protein purified and identified respectively by Ni-NTA agarose and Western-blotting with a molecular weight of 38000, consistent with the theory.Under fluorescent microscope, compared with FITC-NuBCP-9, the fluorescence intensity of FITC-NuBCP-9-r8 significantly increased, indicated that eight polyarginine?R₈? can efficiently across the cell membrane. Compared with biotin-A10/NuBCP-9-r8 group, EMSA results showed the anti-PSMA aptamers targeting autophagic and proapoptotic system presented obviously hysteretic bands, however, BSA, biotin-A10,NuBCP-9-r8 group did not change obviously. The results indicated that the fusion protein SA-ECD could effectively bind biotin labeled anti-PSMA aptamers and NuBCP-9-r8 peptides and successfully constructed the anti-PSMA aptamers targeting autophagic and pro-apoptotic system. The results of cell viability detected by CCK-8 showed A10 targeting system had significant difference on prostate cancer cells?P<0.01?. The corresponding apoptosis percentage of targeting system group from 0 to 40ug/m L was?4.6 ± 1.53?%,?6.98 ± 1.29?%,?16.60 ± 1.30?%,?42.03 ± 1.42?% and?70.66 ± 1.39?% respectively. Except the 5ug/m L group, the remaining groups compared with the control group,the difference was statistically significant?P<0.01?. Trypan blue assay identificated of cell viability, compared with the control group, the cell viability of LNCa P, C4-2, PC-3 and He La in SA-ECD group reduced?P<0.05?, while in A10-NuBCP-9-r8 group did not change obviously?P>0.05?, the cell viability of LNCa P and C4-2 in targeting system group significantly reduced?P<0.01?, showed that this targeting system had a lethal effect on PSMA?+?prostate cancer; Morphological results further prove the above conclusions; Acridine orange and Western blotting analysis showed that targeting system could both induce autophagy and promote apoptosis for LNCa P cells at a certain concentration range.Conclusion: The recombinant plasmid p ET30a-SA-ECD was constructed and the SA-ECD fusion protein was expressed and purified successfully. The preliminary results confirmed that anti-PSMA aptamers targeting autophagic and pro-apoptotic system is specifically cytotoxic to PSMA positive prostate cancer cells, which lay substantial foundation for exploring novel ways to treat prostate cancer.