Gold nanoparticle-based 2′-O-methyl modified DNA probes for breast cancerous theranostics
Introduction
Breast cancer is leading cause of cancer death in women worldwide [1]. Recently, many kinds of therapies have been used for treatment of breast cancer, in which the most successful treatments have been chemotherapy [2], [3] and radiotherapy [4], [5], [6]. However, these therapies are often accompanied by side effects [7], [8]. To alleviate these side effects, many new therapies [9], [10], [11] have been tried for therapeutic breast cancer, of which miRNA-mediated gene silence is an evolving therapeutic strategy [12], [13], [14], [15], [16].
MiRNAs are a class of small non-coding RNAs that modulate protein expression by binding to complementary or partially complementary target mRNAs and thereby targeting the mRNAs for degradation or translational inhibition [17], [18], [19]. MiRNA is a promising endogenous stimulus, as well as a potential therapeutic target because it shows considerable differential expression between the malignant tissues and their normal counterparts [20]. Especially, emerging evidence demonstrates an important role of miRNAs in regulating diverse cellular processes including differentiation, proliferation, apoptosis, metabolism and signal transduction pathways [21], [22], [23]. Individual miRNAs engage numerous mRNAs targets, often encoding multiple components of complex intracellular networks. Thus, the manipulation of miRNAs expression or function can have profound impact on cellular phenotypes [24]. Hence, inhibiting the function of miRNAs by antisense oligonucleotide (that is, antimiRs) is a potential therapeutic strategy, which may yield patient benefits unobtainable by other therapeutic approaches [25], [26], [27]. In recent years, there has been increasing efforts in exploiting antimiRs. For example, Mo et al. transfected antisense oligonucleotide of miRNA-21(antimiR-21) in breast cancer cell and revealed that miRNA-21 is overexpressed in breast tumour tissues and antimiR-21 inhibits both cell growth in vitro and tumour growth in vivo [28]. Ju et al. proposed molecular beacon (MB) conjugated to multifunctional SnO2 nanoparticles with a disulfide linkage using folic acid for cell-specific delivery for imaging and inhibiting intracellular miRNAs-21 [29]. Recently, a novel antimiRs therapeutic platform that targets the acidic microenvironment of tumours has been described, which deliver a peptide nucleic acid (PNA) modified antimiR-155 by pHLIP (low pH-induced transmembrane structure) to inhibit miR-155 in a mouse model of lymphoma and delay tumour growth [30]. Conde et al. used gold-nanobeacon as a theranostic probe for gene specific silencing [31], [32]. MiRNA therapeutics may be superior over other therapies (e.g., single proteins or small molecules) as a miRNA can potentially regulate complex biological processes [33]. miRNA-mediated gene silence had advantages of stability and targeting delivery without potential side effects [34]. However, an ideal miRNA inhibitor platform would display the following properties: high affinity to target genes; low toxicity; high specificity; resistance to exonucleases; relatively low cost for synthesis and the ability to enter cells without use of transfection agents [35].
To satisfy the above criteria, herein, we developed gold nanoparticle-based 2′-O-methyl modified DNA probes (AuNP-2′-OMe-DNA probes) for miRNA-21 detection and inhibition in breast cancer cells. To make sure the better detection and therapeutic effect, antimiR-21 strands are fully modified by 2′-O-methyl to increase binding affinity with tumour gene and improve stability and resistance to exonucleases to prolonged inhibition of miRNAs [36]. As shown in Scheme 1, Au NPs are functionalized with miRNA-21 inhibitors that are hybridized with fluorophore-labeled DNA molecules named “flares”, miRNA-21 inhibitors are 2′-O-methyl modified antisense oligonucleotide that are complementary to the mature miRNA-21 to suppress its function, resulting in cells growth inhibition and apoptotic cells death [27]. On the contrary, abnormal expression miRNA-21 in breast cancer cells may function as an oncogene by blocking expression of critical apoptosis- related genes to promote the growth of cancer cells [27]. In the absence of targets, the close proximity of the fluorophore to the AuNP surface leads to quenching of the fluorescence. However, when a target miRNA-21 binds to the antisense oligonucleotide, the concomitant displacement of the flare can be detected as a corresponding increase in fluorescence and the function of miRNA-21 can be inhibited to realize diagnostic and therapeutic breast cancer cells. (Scheme 1).
Section snippets
Materials and instruments
Trisodium citrate was obtained from Sinopharm Chemical Reagent Co., Ltd. (China). Chloroauric acid (HAuCl4·4HO2) was obtained from Shanghai Chemical Reagent Company (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2-diphenyltetrazolium bromide (MTT) and fetal bovine serum (FBS) were purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Deoxyribonuclease I (DNase I) was purchased from Sangon Biotechnology Co., Ltd (Shanghai, China). Loading buffer was purchased from TaKaRa Bio Inc.
Results and discussions
AuNP-2′-OMe-DNA probes were designed using 13 nm AuNP, since the size particle is an efficient quencher, facile surface modification and does not efficiently scatter visible light [39], which is important for designing optical probes with minimal interference. The TEM image of the Au NPs reveals that the size is uniform (Fig. S1) and UV absorption spectra indicated that the maximum absorption of the AuNP is at 518 nm and that it is red-shifted to 524 nm for the AuNP-2′-OMe-DNA probes (Fig. S2),
Conclusion
In conclusion, we designed a AuNP-2′-OMe-DNA probes for detecting and inhibiting miRNA-21 in breast cancer cells to realize breast cancer diagnostic and therapeutic. 2′-O-methyl was employed to improve stability, increase binding affinity to target strands and enhance the therapeutic effects. AuNP was used as vector to deliver the probes into live cells without use of additional transfection agents, which meet requirement of the ideal miRNA detector and inhibitor. The results demonstrated that
Acknowledgments
This work was supported by the Key Project of the National Natural Science Foundation of China (21735002), the Foundation for Innovative Research Groups of NSFC (21521063), the National Natural Science Foundation of Hunan Province (2017JJ2039), and the Fundamental Research Funds for the Central Universities
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