Application Of Functional Nucleic Acid Probes And MnO₂ Nanosheets In The Treatment Of Breast Cancer

Breast cancer has become the biggest threat to the life and health of Chinese women,the incidence of increasing year by year.Early detection and precision treatment can improve the treatment of breast cancer patients.Functional nucleic acids such as deoxyribozyme and aptamer are widely used in the field of life science.In this paper,two miRNA-responsive nucleic acid functional probes were designed to detect or respond to breast cancer biomarker miRNA-21.Based on the design of the first probe,another DNAzyme/substrate probe was designed,and manganese dioxide nanosheets were used as drug delivery units to construct a composite nanoprobe drug delivery system.For anticancer research.Specific research contents are as follows:1.G-quadruplex-deficient precursor hairpin probes for ultra-low backgroundDesign of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics.Herein,we engineered a miRNA-21 responsive G-quadruplex-deficient precursor hairpin probe（PHP）to achieve dual-mode detection of fluorescent signal and colorimetric signal.Due to lack of complete G-quadruplex sequence,PHP becomes shorter in length,lower background signal and less interference.Based on the polymerase-driven amplification mechanism,in the presence of miRNAs,two simultaneous amplification reaction processes will occur in PHP:miRNA-based amplification process and endogenous amplification process along the 3’end.Due to the positional difference between the starting points of the two amplification processes,the orderly and efficient occurrence of the two amplification processes can be achieved.Based on an interesting concept,PHP can achieve high detection performance with only simple amplification cycles.In such a way,the detection limits for fluorescence and colorimetry were 2.93 f M and 8.81 f M,which would cover most of clinical qualitative and quantitative needs.Thus,the accurate quantitative and visual miRNA detection technology based on PHP is beneficial to carry out extensive disease screening and treatment monitoring in various complex occasions.2.Construction of MnO₂nanosheetsTetramethylammonium hydroxide（TMA·OH）and manganese chloride tetrahydrate（MnCl₂·4H₂O）were be used to synthesize MnO₂ nanosheets under the catalysis of hydrogen peroxide（H₂O₂）to characterize the obtained nanomaterials,and the results also proved that the synthesized materials had uniform particle size,good dispersion,stable structure,good biological safety,and responsive degradation of glutathione（GSH）.3.Nanomaterials based on MnO₂ nanosheets induce C-myc gene silencing for cancer therapyUsing manganese dioxide nanosheets（MnO₂-NS）as vectors,we modified the DNA sequence on its surface and downregulated the antisense oligonucleotide sequence（ASO）of the C-myc gene to construct a nanosystem for tumor treatment（DNAzyme-Locker-Susbstrate-MnO₂）.In this study,we established a bisensitive nanotherapeutic platform for miRNA-21 and glutathione（GSH）based on the DNAzyme amplification strategy for controlled release of drugs.MnO₂ nanosheets are responsible for delivering Locker DNA-locked DNAzyme and substrate DNA into cells.After entering the cell,reduced glutathione（GSH）reduces the MnO₂ nanosheet to Mn²⁺,and subsequently releases the DNA module.The highly expressed miRNA-21 in breast cancer cells binds to Locker DNA through a strand displacement reaction,activating DNAzyme.DNAzyme then cleaves the substrate DNA at cofactor Mn²⁺,releasing the antisense oligonucleotide sequence（ASO）that downregulates the C-myc gene,and when a large amount of substrate DNA is added,a target miRNA results in the release of a large number of antisense oligonucleotide sequences（ASO）.Cell uptake experiments showed that the DNAzyme-Locker-Susbstrate-MnO₂ nanosystem could be effectively taken up by 4T1cells（mouse breast cancer cells）,showed high cell migration inhibitory activity against4T1 cells,and proved that it downregulated C-myc protein expression by immunoblotting.Therefore,the results of this study suggest that the composite nanosystem we designed can induce the silencing of the C-myc gene in breast cancer cells,thereby providing a very promising platform for tumor-targeted therapy.