Ultrasound Combined With Biosynthetic Nanobubbles Mediated Gene Editing Reduces Breast Cancer Metastasis

Objective: Through Ultrasound combined with Biosynthetic nanobubbles(BNBs)to mediate gene editing,down-regulate the expression of N-cadherin(N-cad),a molecule related to Epithelial mesenchymal transformation(EMT),was used to preliminarily explore the effect of this factor on the invasion and metastasis of breast cancer 4T1 cells.Methods: The first part: Cultivation of Halo bacteria and extract BNBs from it,BNBs was characterized by transmission electron microscope and particle size potential analyzer.The second part: Preliminary verification of the feasibility of ultrasound combined with BNBs to mediate gene editing.Firstly,cationic nanobubbles were prepared by incubating BNBs with cationic polymer Polyethylenimine(PEI),and then co-incubated with plasmid DNA(carrying sg RNA which targeted EGFP)to prepare BNBs-PEI-DNA transfection complex.Secondly,it is divided into three groups(Blank group、-US group and +US group)for gene transfection,delivering sg RNA to 4T1 cells which stably expressing Cas9 protein and EGFP.The effect of ultrasound combined with BNBs on promoting gene delivery was observed,and the cell activity after transfection was detected by CCK-8 assay,the monoclonal cells that might succeed in targeting were selected by flow sorting and limited dilution method.Finally,the genomic DNA of targeted monoclonal cells was verified by T7 EI and Sanger sequencing.The third part:with the experimental basis of the previous part,determined the functional target gene,namely CDH2.First of all,the BNBs-PEI-DNA transfection complex we prepared was used to transfect with the same group as above(here,the sg RNA was targeted to CDH2),to observe the delivery effect,and the cell activity after transfection was detected by CCK-8 assay.The monoclonal cells which may be successful in targeting were selected by flow sorting.Then the molecular and protein level verification of monoclonal cells were by T7 EI,Sanger sequencing,and Western Blot.Finally,the cells with CDH2 gene knockout were obtained for morphological observation and functional verification in vitro and in vivo.In vitro,Wound healing and Transwell migration test were used to evaluate the aggressiveness of tumor.In vivo,WT and two successful CDH2 gene knockout monoclonal cells were planted,and the general condition and lung metastasis of mice were observed.Results: 1.BNBs was successfully prepared,the morphology is fusiform,the hydrated particle size is(259.90 ±3.09)nm,and the electric potential is(-21.63 ±0.97)m V.2.The transfection complex BNBs-PEI-DNA was prepared.It was observed that the particle size of the material increased to(386.90±9.15)nm,and the potential changed from negative to positive to(17.90±2.39)m V.The results of gene transfection showed that the transfection efficiency of-US group and +US group was(10.12 ±1.31)% and(25.23 ±2.04)%.The transfection efficiency of +US group was significantly higher than-US group(P<0.05).There was no significant difference in cell activity among the three groups(P>0.05).Subsequently,the monoclonal cells were successfully isolated,and the results of fluorescence intensity,T7 EI and Sanger sequencing also showed positive results.It was confirmed that this method can be used for gene editing and successfully obtained EGFP-knockout cells.3.In the experiment of targeted CDH2,the transfection efficiency of-US group and +US group was(14.63 ±1.50)% and(29.33 ±1.45)%.The transfection efficiency of +US group was significantly higher than-US group(P<0.05).There was no significant difference in cell activity among the three groups(P>0.05).Subsequently,the monoclonal cells were successfully separated by flow sorting,and the positive monoclonal cells were also founded in the results of T7 EI,Sanger sequencing and WB.After the CDH2-knockout cell lines was obtained,the morphological changes were observed.Founded that compared with WT,the morphology of CDH2 knockout cells was slightly rounded,the antennae decreased,the edges were slightly blunt,and the intercellular adhesion was enhanced.The results of Wound healing and Transwell migration test also showed that the migration ability of CDH2-knockout cell lines was significantly lower than WT(P<0.05).In vivo verification,it was observed that the number of metastatic pulmonary nodules in the experimental group after CDH2 knockout was significantly lower than that in the WT group,whether it is an orthotopic tumor model or a tail vein model(P<0.05).Conclusion: Ultrasound combined with BNBs can be used for gene editing to knock out specific genes to obtain corresponding monoclonal cell lines.After knockout of CDH2 gene,the expression of N-cad was down-regulated and the metastatic ability of 4T1 tumor cells decreased,suggested that it may promotes Mesenchymal epithelial transition(MET),and plays a certain role in inhibiting tumor invasion and metastasis,improving prognosis.