Simultaneous Down-Regulation of Intracellular MicroRNA-21 and hTERT mRNA Using AS1411-Functionallized Gold Nanoprobes to Achieve Targeted Anti-Tumor Therapy
Abstract
:1. Introduction
2. Materials and Methods
2.1. Preparing Au NPs and Au Nanoprobes
2.2. Evaluating the Amount of DNA Duplexes Bound on Each Au Nanoprobe
2.3. Determining the Fluorescence Response of Au Nanoprobes to Target DNA
2.4. Determining the Fluorescence Response of Au Nanoprobes to Intracellular MicroRNA-21 and hTERT mRNA
- (i)
- Cell lysate analysis: Cell lysates were obtained by breaking down MCF-7 cells (1 × 106) using an ultrasonic disruptor. Au nanoprobes (1.5 nM) were incubated with the freshly prepared cell extracts at 37 °C for 4 h. The fluorescence intensity levels of the experimental systems were determined by using the F-7000 spectrofluorometer (Hitachi, Japan) with excitation wavelengths of 530 and 630 nm.
- (ii)
- In situ fluorescence imaging: MCF-7 or HeLa cells (0.4 mL, 1 × 106 mL−1) were, respectively, seeded in a 20 mm glass-bottom confocal dish. After 24 h, Au nanoprobes (1.5 nM) were incubated with cells for 4 h. Then, cells were washed with PBS thrice and observed by LSM880 confocal laser scanning microscopy (CLSM, Zeiss, Jena, Germany). The fluorescence signals of Cy3 and Cy5 of Au nanoprobes responsive to hTERT mRNA and microRNA-21 were excited with wavelengths of 543 and 633 nm, respectively.
2.5. Quantifying the Uptake of Au Nanoprobes in Cancer Cells
2.6. Analyzing the Intracellular hTERT mRNA and microRNA-21 Level
2.7. Analyzing the Intracellular hTERT Activity
2.8. Determining the Pro-Apoptosis Effect and In Vitro Cytotoxicities of Au Nanoprobes
2.9. In Vivo and Ex Vivo Fluorescence Imaging and Quantifying the Accumulation of Au Nanoprobes in Tumors
2.10. In Vivo Anti-Tumor Study
2.11. Statistical Analysis
3. Results and Discussion
3.1. Design Mechanism of Au Nanoprobes
3.2. Characterization of AuNPs and Au Nanoprobes
3.3. Evaluation of the Amount of DNA Duplexes Bound on Each Au Nanoprobe
3.4. In Vitro Response of Au Nanoprobes to hTERT mRNA and microRNA-21-Related Target DNA
3.5. In Situ Fluorescence Imaging of Intracellular hTERT mRNA and microRNA-21 Using Au Nanoprobes and Evaluating the Intracellular Amount of Au Nanoprobes
3.6. Analysis of the Expression Level of hTERT mRNA and microRNA-21 as Well as hTERT Activity in Cancer Cells Treated with Au Nanoprobes
3.7. Studies of the Pro-Apoptosis Effect and In Vitro Cytotoxicity of Au Nanoprobes
3.8. In Vivo and Ex Vivo Imaging of Au Nanoprobes
3.9. In Vivo Targeted Anti-Tumor Therapy
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Conflicts of Interest
References
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Ji, Q.; Yang, Q.; Ou, M.; Hong, M. Simultaneous Down-Regulation of Intracellular MicroRNA-21 and hTERT mRNA Using AS1411-Functionallized Gold Nanoprobes to Achieve Targeted Anti-Tumor Therapy. Nanomaterials 2024, 14, 1956. https://doi.org/10.3390/nano14231956
Ji Q, Yang Q, Ou M, Hong M. Simultaneous Down-Regulation of Intracellular MicroRNA-21 and hTERT mRNA Using AS1411-Functionallized Gold Nanoprobes to Achieve Targeted Anti-Tumor Therapy. Nanomaterials. 2024; 14(23):1956. https://doi.org/10.3390/nano14231956
Chicago/Turabian StyleJi, Qinghong, Qiangqiang Yang, Mengyao Ou, and Min Hong. 2024. "Simultaneous Down-Regulation of Intracellular MicroRNA-21 and hTERT mRNA Using AS1411-Functionallized Gold Nanoprobes to Achieve Targeted Anti-Tumor Therapy" Nanomaterials 14, no. 23: 1956. https://doi.org/10.3390/nano14231956
APA StyleJi, Q., Yang, Q., Ou, M., & Hong, M. (2024). Simultaneous Down-Regulation of Intracellular MicroRNA-21 and hTERT mRNA Using AS1411-Functionallized Gold Nanoprobes to Achieve Targeted Anti-Tumor Therapy. Nanomaterials, 14(23), 1956. https://doi.org/10.3390/nano14231956