The Construction And Targeting Therapy Of Synthetic Biological Devices Mediated By The HTERT Promoter In Bladder Cancer Cells

Background and objectives: In order to explore new methods for the treatment of bladder cancer,this study adopted the concept of synthetic biology,designed and synthesized regulatory elements,and observed its impact on the development of malignant bladder tumour.Methodology: GAL4 vectors driven by h TERT promoter and CRISPR/Cas9 system carriers driven by the upstream activating sequence(UAS)were constructed,and co-transfected into cells.Dual luciferase reporter assays were used to verify the expression activity of h TERT promoter in cell lines HFF,5637,and T24,and the regulated effect of GAL4/UAS binary system in each cell line.Real-time quantitative PCR(q PCR)was used to detect the expression of HRAS gene.Sanger sequencing was used to detect whether the target sequences of HRAS gene was cut in negative control and experimental group cells.Cell proliferation,apoptosis,migration and invasion were detected by CCK-8 assay,flow cytometry,cell scratch scratches and transwell assay respectively.Results: The dual luciferase assay showed that the h TERT promoter had activation ability only in T24 and 5637 cells but not in HFF cells.In the bladder cancer cells T24 and 5637,the fusion protein GAL4-P65 had a significantly higher activation effect on the downstream activation sequence UAS than GAL4-VP64.q PCR showed that the regulatory elements knocked down the expression of HRAS gene in T24 cells and 5637 cells significantly.Sanger sequencing showed that the HRAS gene was genetically excised by regulatory elements in bladder cancer cells T24 and 5637.CCK-8 assay,flow cytometry,cell scratch assay,and transwell assay demonstrated that the artificial regulatory components had no significant effect on the proliferation,apoptosis,migration and invasion of HFF cells,but on bladder cancer cells T24 and 5637 promoted apoptosis and inhibited cell proliferation,migration and invasion.Conclusions: The regulatory system consisting of enhanced h TERT promoter,GAL4/UAS system and CRISPR-Cas9 tool selectively inhibited the expression of HRAS in T24 cells and 5637 cells.Meanwhile,the regulatory promoted cell apoptosis and inhibit cell proliferation,migration and invasion in bladder cancer.