Dna Catalyzed Hairpin Assembly Technology Applied In MicroRNA-21 Detection And Killing Effect On Cervical Cancer Cells

Background and purpose:Cervical cancer is a serious disease affecting the health of woman.Studies showed that micro RNA-21(miR-21)is over expressed in He La cells and miR-21 gene adjacent to human papillomavirus(HPV)integration site may be related to its over expression level in cervical cance.Therefore,it is of great significance to detect miR-21 in He La cells.In addition,vascular endothelial growth factor(VEGF)is closely related to the occurrence,development and prognosis of cervical cancer,which is an important target for the treatment of cervical cancer.As a tool of gene therapy,it is very important to develop an efficient si RNA delivery system.Based on DNA self-assembly technology,this thesis realized the isothermal amplification detection and in situ imaging of miR-21 in He La cells.Meanwhile,in situ generation of VEGF si RNA is achieved for gene therapy of cervical cancer,which provides a new research direction for the treatment of cervical cancer.Methods:The designed enzyme-free DNA circuit consists of three kinds of DNA hairpins(H1,H2/PS and H3),in which miR-21 initiates the hairpin assembly reaction to achieve target cycling signal amplification and construct "DNA nanowheels" structures for high sensitivity and specificity for detection of miR-21.The feasibility of this strategy was verified by polyacrylamide gel electrophoresis(PAGE)and Atomic force microscope(AFM).Fluorescence kinetics and fluorescence spectra were obtained to demonstrate the relation between fluorescence intensity and concentration of miR-21,as well as the specificity of miR-21 triggered the enzyme-free DNA circuit.Confocal laser scanning microscopy imaging(CLSM)was used for bioimaging analysis of the expression and distribution of miR-21 in living cells.The sense sequence and antisense sequence of VEGF si RNA were embedded in hairpin structures,overexpressed miR-21 in He La cells triggered the catalytic hairpin assembly reaction,generating "DNA nanowheels" structures containing a large number of VEGF si RNA.By virtue of the endogenous enzyme(Dicer)processing,VEGF si RNA were cut off to specifically inhibit the expression of VEGF gene in He La cells.Real-time fluorescent quantitative PCR(q RT-PCR)and western blot(WB)assays demonstrated the effect of gene silencing.Flow cytometry and CCK-8 assays indicated in situ generation of VEGF si RNA has the excellent killing capacity for He La cells.Furthermore,the therapeutic effect of enzyme-free DNA circuit in vivo was examined by constructing subcutaneous xenograft tumor of cervical cancer He La cells in nude mice.Results:(1)PAGE results showed that miR-21 successfully triggered the self-assembly of DNA hairpins and generated DNA structures with large molecular weight.AFM results showed that compared to the mixture of DNA hairpins without adding miR-21,the introduction of miR-21 generated a large number of monodisperse spherical "DNA nanowheels" structures.All those results verified the feasibility of miR-21-responsive enzyme-free DNA circuit.Fluorescence kinetics and fluorescence spectra results showed the fluorescence intensity of the operation of enzyme-free DNA circuit was dependent on the increase concentration of miR-21.CLSM imaging results showed that He La cells transfected with DNA hairpins had strong fluorescence signal,while no fluorescence recovery was observed in NR-circuit group and anti-miR-21 treated He La cells,indicating the specificity of miR-21 triggered the enzyme-free DNA circuit.Additionally,this system was further applied to in situ imaging of miR-21 in different living cells(Hep G2 cells and L-02 cells).The results showed that the fluorescence recovery was observed in Hep G2 cells with high expression of miR-21,but no fluorescence recovery in L-02 cells with low expression of miR-21,indicating the universality of this enzyme-free DNA circuit.(2)q RT-PCR and WB results showed that in situ generation of VEGF si RNA via miR-21-responsive enzyme-free DNA circuit can effectively inhibit the expression of VEGF m RNA and related protein.Compared to naked si RNA group(the inhibition expression of VEGF m RNA and VEGF protein were 29.22% and 41.89%),the inhibition expression of VEGF m RNA and VEGF protein of DNA circuit were 48.94% and 64.46%,demonstrating the high gene silence efficiency.Meanwhile,the apoptosis experiment showed that in situ generation of VEGF si RNA resulted in higher apoptosis rate(42.40%)compared with the naked si RNA group(30.30%).It can be contributed to the protect effect of DNA circuit in which DNA hairpins can protect si RNA from degradation.CCK-8 assay results showed DNA circuit had the lowest cell proliferation(32.86%),which was consistent with the above results.Furthermore,the results of xenograft tumor experiment showed that DNA circuit had the most obvious inhibitory effect on tumor,and the hematoxylin eosin(H&E)staining of main organs showed no organ toxicity in all groups,indicating that the designed system had excellent biocompatibility and enhanced anti-tumor ability.Conclusion:(1)The proposed enzyme-free DNA circuit has worked as a powerful tool for isothermal amplification detection and in situ imaging analysis of miR-21 in living cells,which exhibits high sensitivity and good specificity.(2)The constructed enzyme-free DNA circuit can protect VEGF si RNA from degradation in the complex tumor microenvironment,exhibiting excellent biological stability to increase the gene silencing efficiency and apoptosis rate.(3)Furthermore,the established method was applied to He La cell xenograft of cervical cancer,which effectively inhibited the growth of the cervical carcinoma cells.Overall,this strategy provided a new research for the application of DNA nanotechnology.