Design,Construction And Function Test Of P53 Mutant Tumor-specific Interference Gene Circuits

Objective:Use synthetic biology technology to design and construct p53 mutant tumor-specific intervention gene circuits,and to determine experimental therapeutic effect and specificity of the gene circuits on p53 mutant tumor.Methods:The aptazyme which can specifically bind to wild type p53 protein was constructed by the method of vector construction,and the activity and specificity of p53 aptazyme in cells were verified by Dual-Luciferase Reporter assay.The Cre-p53 aptazyme gene circuit and CRISPR-p53 aptazyme gene circuit were constructed by the method of vector construction.Fluorescence quantitative PCR and Western blot assays were used to detect the expression of DTA(diphtheria toxin part A,diphtheria toxin A fragment)induced by Cre-p53 aptazyme gene circuit and p53 induced by CRISPR-p53 aptazyme gene circuit in p53 mutant tumor cell lines.CCK-8 assay and flow cytometry were used to detect the effect of Cre-p53 aptazyme gene circuit and CRISPR-p53 aptazyme gene circuit on tumor cell proliferation and apoptosis.Nude mouse tumor formation experiment was used to detect the effect of Cre-p53 aptazyme gene circuit and the CRISPR-p53 aptazyme gene circuit on tumor formation in nude mice.Results:Western blot results showed that total p53 was expressed in HFF,293T,HCT116 p53+/+and 5637 cells,while there was almost no expression of p53 protein in HCT116 p53-/-,G361,SCL-1 and Hela cells.The results of dual-luciferase report assay showed that p53 aptazyme could significantly reduce the luciferase activity in p53 wild type cells(HFF,293T and HCT116 p53+/+),but have no significant effect on luciferase activity in wild type p53 deficient cells(HCT116 p53-/-,5637,G361,SCL-1 and Hela).The results showed that p53 aptazyme switch could specifically act on p53 wild type cells and activate the cleavage activity of hammerhead ribozyme.The sequencing results of Cre-p53 aptazyme and CRISPR-p53 aptazyme gene circuits showed that the full-length sequence of the vectors was constructed correctly and the insertion position of p53 aptazyme was accurate.The results of fluorescence quantitative PCR and Western blotting showed that Cre-p53 aptazyme gene circuit could induce significant expression of DTA in wild type p53 deficient cells but in HFF,293T and HCT116+/+cells.In addition,the CRISPR-p53 aptazyme gene circuit could induce significant expression of p53 in wild type deficient cells(5637,G361,SCL-1 and Hela)but in HFF,293T and HCT116+/+cells.CCK-8 and flow cytometry assays showed that Cre-p53 aptazyme gene circuit could significantly inhibit the proliferation and promote apoptosis of HCT116 p53-/-,5637,G361,SCL-1 and Hela cells.Additionally,CRISPR-p53 aptazyme gene circuit could inhibit the proliferation and promote the apoptosis of G361,SCL-1 and Hela cells.The tumor formation results of nude mice showed that Cre-p53 aptamer ribozyme gene circuit could significantly inhibit the growth of HCT116 p53-/-,5637,G361,SCL-1 and Hela tumors.Futhermore,the CRISPR-p53 aptazyme gene circuit could significantly inhibit the growth of G361,SCL-1 and Hela tumors.Conclusions:The p53 aptazyme can effectively sense the p53 protein signals in cells and respond in time to auto-cleavage.The Cre-p53 aptazyme gene circuit and the CRISPR-p53 aptazyme gene circuit constructed with p53 aptazyme as the main component can target the tumor cells with abnormal p53 protein,but have no effect on the cells with normal expression of p53 protein.The function of p53 mutant tumor-specific interference gene circuit constructed with p53 aptazyme as the core is in line with expectations.As a new method,this type of gene circuit has the potential of tumor biotherapy.