Construction Of Triplex Nucleic Acid Gene Regulation System And Study On Tumor Cell Killing

Cancer has become a major problem threatening human life and health around the world.However,there is still no significant breakthrough in the treatment of cancer by current treatment methods.With the improved research on the pathogenesis of cancer,it has been found that the abnormal expression of micro RNAs（miRNAs）was closely related to the occurrence and development of cancer,involving high expression of oncogenic miRNAs and low expression of tumor suppressor miRNAs,which was recognized as promising therapeutic targets for cancer.However,the delivery of a single species of miRNAs has limited regulatory effects on tumor cells.What’s more,current miRNAs-based therapeutic strategies still face the challenges of uncontrollable release and insufficient specificity.Based on this,the thesis introduces the triplex molecular switch into the miRNAs delivery system.By constructing a logic-responsive triplex nucleic acid gene regulation system,the specific identification and controllable release of tumor suppressor miRNAs and oncogenic miRNA inhibitor at the molecular level can be conducted to improve the tumor inhibition selectivity;Then triplex switch and anti-tumor drugs are further loaded on nanomaterials,which applying to the discriminative killing of drug-resistant and sensitive tumor cells.Specifically,it includes the following aspects:1.Construction and characterization of miRNAs-mediated logical-responsive triplex gene regulatory systemTo solve the issue of uncontrolled release of miRNAs in“therapeutic gene”,it was developed a novel gene regulation system based on miRNAs stimulus-responsive clamp-like triplex switch（CTS）formed by Watson-Crick and Hoogsteen hydrogen bonds.In the presence of miRNA-155,the CTS could be specifically opened,showing a conformational change from triplex to linear and obviously enhanced fluorescence.The results showed that the system has good selectivity,high sensitivity and stability.In addition,the strategy has wide versatility and flexibility in design,providing the foundation for the further design and application of multifunctional nanostructure assembling systems.2.micro RNAs-mediated logical-responsive triplex gene regulation system for tumor cell killingBased on the previous chapter,the CTS was applied for breast cancer tumor cells to achieve the bidirectional regulation of miRNAs and enhance programmed gene therapy.Compared with“unidirectional”regulation method,CTS showed 1.5 to 2.0 times increased cytotoxicity to MCF-7 cells,and significantly reduced their proliferation and migration abilities.However,CTS had no toxic side effects on normal cells.Therefore,CTS-based strategies provide a new idea for precision medicine in cancer and controlled release of drugs.3.Construction and characterization of a dual-activated MnO₂@TNS/DOX gene regulatory systemTo improve the therapeutic efficacy,we developed a dual-activated gene regulation system MnO₂@TNS/DOX to achieve the controllable release of three drugs.Manganese dioxide nanosheets with high drug loading rate,which acted as both a nanocarrier and quencher,programmably loaded the triplex nanoswitch（TNS）and doxorubicin（DOX）through physical adsorption and electrostatic interaction.The results showed that the system had a large loading capacity,and the TNS and DOX amounts of 1μg MnO₂ nanosheets were 12.1 pmol and 4.78nmol,respectively.In the presence of GSH,TNS and DOX were simultaneously released,and their release amounts were both as high as 90%within 17 min.For TNS,in the presence of the P-gp m RNA（m RNA of encoding P-glycoprotein）,the switch was specifically recognized by the target to induce its conformational reorganization,and promoted the switch to open for inducing fluorescence changes,thereby realizing the controllable release of various drugs.In addition,the system showed high stability in different medium.Therefore,this system is an ideal intracellular dual stimuli-responsive gene regulation system,providing a new tool for precise and personalized medical applications in cancer.4.Dual-activated MnO₂@TNS/DOX gene regulation system for differential killing of resistant and sensitive tumor cellsBased on the previous section,to improve the problem of multidrug resistance in cancer chemotherapy,the system was applied for drug-resistant breast cancer cells,which achieved distinguishingly killing of DOX-resistant and sensitive tumor cells.The results showed that the system could be activated by the highly expressed GSH in tumor cells,and released TNS and DOX;for the target cells,TNS was specificity activated by P-gp m RNA,and while P-gp m RNA was silenced,let-7a mimic was continuously replenished,thereby achieving high-specificity and high-contrast imaging of tumor cells.With the programmed release of the three drugs,the drug resistance of tumor cells was greatly improved,and bidirectional regulation of genes was finally achieved.Compared with the sensitive cells,the system had a more significant inhibitory effect on the resistant tumor cells.The system achieves logically controlled release of different drugs in a specific space by a programmed release,which is expected to be broadly used in the field of precise and controlled drug release.