microRNAs In Cancer Cells Research Articles

Three-in-one DNA nanowheels for simultaneous tumor regression and drug resistance prevention in breast cancer model

Herein, we proposed novel three-in-one DNA nanowheels with simultaneous chemo and gene therapy to treat tumor, especially to prevent simultaneous drug resistance, which could be disassembled via a cascaded hybridization reactions triggered by the highly expressed microRNA in cancer cells for smart and efficient cancer therapy. Typically, with breast cancer as a model, microRNA 21 could trigger the self-disassembly of DNA nanowheel 1 via hybridization with a specially designed oligonucleotide (anti-microRNA 21) in DNA nanowheel 1, releasing another special oligonucleotide (Contact sequence) to trigger the self-disassembly of DNA nanowheel 2 with releasing of a special oligonucleotide (anti-Contact sequence) to trigger the self-disassembly of DNA nanowheel 1 cyclically, and thus the cascaded hybridization reactions with three-in-one anti-cancer functions could be generated based on three main therapeutic effects via releasing doxorubicin to inhibit macromolecular biosynthesis, antisense oligonucleotide of microRNA 21 to activate the apoptotic cell pathway and antisense oligonucleotide of MDR1 to prevent the drug resistance respectively. As expected, the proposed method showed improved therapeutic efficacy on the cancer cells with about 80% apoptosis ratio, especially on the drug resistant cancer cells with about 75% apoptosis ratio, compared with that in the conventional anti-cancer systems of about 70% on cancer cells and below 40% on drug resistant cancer cells, respectively. Most importantly, this strategy opened the door for generation of complex functional DNA-based structures for target triggering drugs releasing system combining with chemo- and gene-therapy to generate tumor regression and prevent drug resistance with an optimized therapeutic efficacy, providing a new avenue for efficient cancer treatment, especially drug resistant cancers.

Visual and Electrochemical Detection of let-7a: A Tumor Suppressor and Biomarker.

Let-7a, a type of low-expressed microRNAs in cancer cells, has been investigated as a promising biomarker and therapeutic target for tumor suppression. Developing simple and sensitive detection methods for let-7a is important for cancer diagnosis and treatment. In this work, the hybridization chain reaction (HCR) was initiated by let-7a via two hairpin primers (H1 and H2). After the HCR, the remaining hairpin H1 was further detected by lateral flow assay (LFA) and electrochemical impedance spectroscopy. For LFA, biotin-modified H1(bio-H1) and free H2 were used for HCR. With the decrease of let-7a concentration, the color of T line gradually increased. As for electrochemical methods, the H1'-AuNP-modified electrode was used for detection of bio-H1 based on the difference of impedance (ΔRct) detected without and with different concentrations of let-7a participating in the HCR. This method could detect let-7a in the range of 10.0 fM and 1.0 nM with detection limits of 4.2 fM.

Dual-potential ratiometric electrochemiluminescence microRNA bioassay based on inner reference probe and catalytic hairpin assembly

Dual-signal ratiometric biosensors have drawn great attention due to their good reproducibility, reliability, and anti-interference ability in sensitive biosensing. Herein, we propose a dual-potential ratiometric electrochemiluminescence (ECL) biosensor based on an internal reference probe and catalytic hairpin assembly (CHA) signal amplification strategy. The cathodic ECL signal of Au-deposited graphitic carbon nitride nanosheets (Au-g-C3N4) was selected as the inner reference, while the anodic ECL response of luminol-H2O2 in solution was used as the reporter signal. After target recognition, Pt nanocluster-labeled DNA strands were introduced onto the surface of the electrode by DNA hybridization, effectively accelerating the production of reactive oxygen species (O2•–) and greatly enhancing the reporter signal with high catalytic activity. The ratio of ECL intensity between the enhanced anodic signal and stable cathodic signal varies with a good linear relationship to the logarithm of microRNA concentration during the detection procedure. Assisted by target-induced CHA signal amplification, this ratiometric ECL biosensor displayed favorable performance for the sensitive detection of microRNA-21 with build-in correction against circumventing fluctuations. The expression level of microRNA-21 in cancer cells is evaluated with favorable reliability and reproducibility and displays great potential and priority in complex biosensing and life assays.

Dendronized Polymeric Biomaterial for Loading, Stabilization, and Targeted Cytosolic Delivery of microRNA in Cancer Cells

Dendronized Polymeric Biomaterial for Loading, Stabilization, and Targeted Cytosolic Delivery of microRNA in Cancer Cells

Localized plasmonic sensor for direct identifying lung and colon cancer from the blood

The tissue inhibitor of metalloproteinases-1 (TIMP-1) protein can regulate the expression of certain proteases and microRNAs in cancer cells, and it is highly possible to diagnose cancers through analyzing the expression of TIMP-1 on exosomes. However, it is still a great challenge to obtain reliable physiological information on TIMP-1 by label-free method from exosomes in plasma. Here, we designed a porous-plasmonic SERS chip functionalized with synthesized CP05 polypeptide, which can specifically capture and distinguish exosomes from diverse origins. The SERS chip can accurately locate the plasmon in TIMP-1 protein to analyze the discrepancy of related fingerprint peaks of different exosomes. Based on the designed SERS chip, we successfully distinguished the lung and colon cancer cell-derived exosomes from normal exosomes at the single vesicle level by unique Raman spectroscopy and machine learning methods. This work not only provides a practical SERS chip for the application of Raman technology in human tumor monitoring and prognosis, but also provides a new idea for analyzing the feature of exosomes at the spectral level.

Simultaneous Imaging of Dual microRNAs in Cancer Cells through Catalytic Hairpin Assembly on a DNA Tetrahedron.

Accurate detection and imaging of tumor-related microRNA (miRNA) in living cells hold great promise for early cancer diagnosis and prognosis. One of the challenges is to develop methods that enable the identification of multiple miRNAs simultaneously to further improve the detection accuracy. Herein, a simultaneous detection and imaging method of two miRNAs was established by using a programmable designed DNA tetrahedron nanostructure (DTN) probe that includes a nucleolin aptamer (AS1411), two miRNA capture strands, and two pairs of metastable catalytic hairpins at different vertexes. The DTN probe exhibited enhanced tumor cell recognition ability, excellent stability and biocompatibility, and fast miRNA recognition and reaction kinetics. It was found that the DTN probe could specifically enter tumor cells, in which the capture strand could hybridize with miRNAs and initiate the catalytic hairpin assembly (CHA) only when the overexpressed miR-21 and miR-155 existed simultaneously, resulting in a distinct fluorescence resonance energy transfer signal and demonstrating the feasibility of this method for tumor diagnosis.

Construction of a Structure-Switchable Toehold Dumbbell Probe for Sensitive and Label-Free Measurement of MicroRNA in Cancer Cells and Tissues.

MicroRNAs (miRNAs) play multiple crucial roles in post-transcriptional regulating gene expression, and the abnormal expression may induce various human diseases. Herein, we demonstrate the construction of a structure-switchable toehold dumbbell probe for sensitive and label-free measurement of microRNA in cancer cells and tissues on the basis of integrating exponential-rolling circle amplification (EXP-RCA) with linear-rolling circle amplification (LRCA). We designed a structure-switchable toehold dumbbell probe with annular and symmetric structure whose either side can hybridize with target miRNA to initiate EXP-RCA, greatly improving the detection sensitivity. Moreover, the dumbbell probe is designed with an appropriate standard free energy (G), and it cannot be activated by mismatched miRNAs, endowing this assay with good specificity. When target miRNA is present, it interacts with the dumbbell probe to activate EXP-RCA via toehold-mediated stand displacement, generating abundant triggers. The resulting triggers and target miRNA can function as primers to initiate LRCA, producing abundant long tandem repeats that can generate a distinct fluorescence signal using SYBR Gold as the indicator. This assay can be carried out homogeneously and isothermally without the requirement for either sophisticated modification/separation steps or any extra primers. It displays ultrahigh sensitivity with a limit of detection of 8.45 × 10-17 M and excellent specificity, and it can differentiate let-7a from its homologous analogues. Moreover, this method can accurately quantify let-7a expression at a single-cell level and can even distinguish the let-7a expression between non-small cell lung cancer patient tissues and healthy person tissues.

Expression Profiles of Estrogen-Regulated MicroRNAs in Cancer Cells.

MicroRNAs play critical roles through their impact on posttranscriptional gene regulation. In cancer, they can act as oncogenes or tumor suppressors and can also function as biomarkers. Here, we describe a method for robust characterization of estrogen-regulated microRNA profiles. The activity of estrogen is mediated by two nuclear receptors, estrogen receptor alpha and estrogen receptor beta, and a transmembrane G-protein coupled estrogen receptor 1. This chapter details how to prepare cells for optimal estrogen response, directions for estrogen treatment, RNA extraction, different microRNA profiling approaches, and subsequent confirmations.

Modulating microRNAs in cancer: Next-generation therapies.

MicroRNAs (miRNAs) are a class of endogenously expressed non-coding regulators of the genome with an ability to mediate a variety of biological and pathological processes. There is growing evidence demonstrating frequent dysregulation of microRNAs in cancer cells, which is associated with tumor initiation, development, migration, invasion, resisting cell death, and drug resistance. Studies have shown that modulation of these small RNAs is a novel and promising therapeutic tool in the treatment of a variety of diseases, especially cancer, due to their broad influence on multiple cellular processes. However, suboptimal delivery of the appropriate miRNA to the cancer sites, quick degradation by nucleases in the blood circulation, and off target effects have limited their research and clinical applications. Therefore, there is a pressing need to improve the therapeutic efficacy of miRNA modulators, while at the same time reducing their toxicities. Several delivery vehicles for miRNA modulators have been shown to be effective in vitro and in vivo. In this review, we will discuss the role and importance of miRNAs in cancer and provide perspectives on currently available carriers for miRNA modulation. We will also summarize the challenges and prospects for the clinical translation of miRNA-based therapeutic strategies.

MicroRNA-Triggered Nanozymes Cascade Reaction for Tumor-Specific Chemodynamic Therapy.

Off-target toxicity and insufficient hydroxyl radicals (. OH) generation limit the further clinical application of nanozymes in chemodynamic therapy (CDT). Herein, we designed and constructed a microRNA-triggered nanozyme cascade platform for enhanced tumor-specific chemodynamic therapy. The nanozyme-based cascade reaction could be triggered successfully by the high expression of microRNA in cancer cells to generate more . OH, thus exhibiting excellent tumor-specific therapeutic performance. Our work provides a new dimension for tumor-specific chemodynamic therapy.

Iridium oxide nanoparticles-based theranostic probe for in vivo tumor imaging and synergistic chem/photothermal treatments of cancer cells

A novel iridium oxide nanoparticles-based probe was developed for in vivo fluorescence imaging of microRNA and synergistic chem/photothermal treatments of tumor. The theranostic nanoprobe was constructed by functionizing iridium oxide nanoparticles with split DNAzyme precursor and chemotherapeutic agent doxorubicin. The split DNAzyme precursor was used for specifically recognizing the target microRNA in cancer cells and then releasing the preloaded fluorescence for detection through DNAzyme cleavage reaction. Because the fluorescence was enhanced by cycle amplification process, the nanoprobe exhibited high sensitivity in the cell-free test. Both in vitro and in vivo fluorescence imaging studies proved the specificity and feasibility of the nanoprobe in tumor imaging. In order to enhance the effectiveness of cancer cell therapy, synergistic chem/photothermal treatments were designed on the nanoprobe. Based on the photothermally controlled drug release and chemotherapeutic treatment of doxorubicin, and the photothermal effect of iridium oxide nanoparticles, the nanoprobe exhibited excellent synergy effect on cancer cell treatment under near-infrared irradiation. In particular, in vivo therapeutic studies proved a great inhibition of the tumor growth. Therefore, this work provided a promising theranostic nanoprobe for both tumor imaging and synergistic treatment of cancer cells.

Photoelectrochemical Biosensor for MicroRNA-21 Based on High Photocurrent of TiO2/Two-Dimensional Coordination Polymer CuClx(MBA)y Photoelectrode.

Conventional photosensitive materials such as TiO2 suffer from restricted absorption in the ultraviolet region, fast recombination of photogenerated electron-hole pairs, and a lack of functional groups for biocoupling, which hinder their application in photoelectrochemical (PEC) biosensing. Herein, a new coordination polymer (CP) based on Cu(I), chloridion, and 4-mercaptobenzoic acid (MBA) has been designed and synthesized (called CuClx(MBA)y). The prepared p-type CuClx(MBA)y exhibits visible-light absorption due to its narrow optical band gap (2.59 eV), and its proper band edge position enables it to form a p-n junction with TiO2. Through layer-by-layer assembling, the photocurrent intensity of the CuClx(MBA)y/TiO2/FTO composite photoelectrode was 3.7-fold higher than that of a TiO2/FTO electrode and 35-fold higher than a CuClx(MBA)y/FTO electrode. The potential enhancement mechanism was discussed, which lies in the contributions of CuClx(MBA)y in enhancing absorption in the visible-light region and boosting the separation of electron-hole pairs of TiO2 by the p-n junction. Furthermore, CuClx(MBA)y nanosheets can realize bioconjugation directly, thanks to its abundant carboxyl groups. The CuClx(MBA)y/TiO2/FTO composite photoelectrodes were applied to develop a sensitive PEC biosensor for microRNA-21 (model target). By subtly exploiting the energy transfer between CuClx(MBA)y and Au nanoparticles (AuNPs), AuNPs served as effective quenchers. In the presence of the target, AuNP-labeled sDNA1 connected to the electrode surface, and thus, a decreased photocurrent was obtained. The proposed biosensor has a low detection limit of 0.29 fM (S/N = 3), good selectivity, and reproducibility. The proposed system was applied to monitor microRNA in cancer cells with satisfying results.

Digital Counting of Biomolecules Using Engineered Functional DNA Superstructures.

There is currently a great need for developing a simple and effective biosensing platform for the detection of single biomolecules (e.g., DNAs, RNAs, or proteins) in the biological, medical, and environmental fields. Here, we show a versatile and sensitive fluorescence counting strategy for quantifying proteins and microRNAs by employing functional DNA superstructures (denoted as 3D DNA). A 3D DNA biolabel was first engineered to become highly fluorescent and carry recognition elements for the target of interest. The presence of a target cross-links the resultant of the 3D DNA biolabel and a surface-bound capturing antibody or DNA oligonucleotide, thus forming a sandwich complex that can be easily resolved using traditional fluorescence microscopy. The broad utility of this platform is illustrated by engineering two different 3D DNA biolabels that enable the quantification of β-lactamase (one secreted bacterial hydrolase) and miR-21 (one overexpressed microRNA in cancer cells) with detection limits of 100 aM and 1 fM, respectively. We envision that the approach described herein will find useful applications in chemical biology, medical diagnostics, and biosensing.

A copper-free and enzyme-free click chemistry-mediated single quantum dot nanosensor for accurate detection of microRNAs in cancer cells and tissues.

MicroRNAs (miRNAs) play key roles in the post-transcriptional regulation of genes, and their aberrant expression may disturb the normal gene regulation network to induce various diseases, and thus accurate detection of miRNAs is essential to early clinical diagnosis. Herein, we develop for the first time a single-quantum dot (QD)-based Förster resonance energy transfer (FRET) nanosensor to accurately detect miRNAs based on copper-free and enzyme-free cycling click chemistry-mediated tricyclic ligase chain reaction (LCR) amplification. We design four DNA probes namely DNA probes 1–4, with DNA probes 1 and 3 being modified with azide (N3) and DNA probes 2 and 4 being modified with dibenzocyclooctyne (DBCO). When target miRNA is present, DNA probes 1 and 2 can proceed via copper-free and enzyme-free click chemistry to generate the probes 1–2 ligation product. Subsequently, DNA probes 3 and 4 can hybridize with the probes 1–2 ligation product to generate the probes 3–4 ligation product. Both the probes 1–2 ligation product and probes 3–4 ligation product can act as the templates to initiate cycling click chemistry-mediated tricyclic LCR amplification whose products can be easily measured by the single-QD-based FRET nanosensor. This assay does not involve any enzymatic reverse transcription, copper catalyst, and ligase enzyme, and it exhibits excellent selectivity, high sensitivity, and the capability of differentiating even single-base mismatches. Moreover, this nanosensor can accurately quantify miRNA-155 even at the single-cell level, and it can distinguish the miRNA-155 expression in tissues of healthy persons and nonsmall cell lung cancer (NSCLC) patients.

Sensitive detection of microRNA-21 in cancer cells and human serum with Au@Si nanocomposite and lateral flow assay

We report a highly sensitive approach for detecting microRNA-21 (miR-21) in cancer cells and human serum by using Au@Si nanocomposite labeled lateral flow assay. The Au@Si nanocomposite was prepared by coating numerous 3–5 nm gold nanoparticles (GNP) on a silica nanoparticle (SiNP) with a diameter of 150 nm and used as colored label on the lateral flow assay for signal amplification. TEM results show there are around 1000 GNPs coated on the SiNP surface. The principle of miR-21 detection is based on on-strip DNA-microRNA hybridization reactions to form DNA-miR-21-DNA-Au@Si complexes, which are captured on the test zone of the lateral flow test strip and produce a visible red band. A thiol-modified detecting DNA probe (Det-DNA) and a biotin-modified capturing DNA probe (Cap-DNA), which are complementary to miR-21, were used to prepare the lateral flow test strips. After systematic optimization, the method can detect a minimum concentration of 1.0 pM miR-21, which is 60 times lower than that of the GNP-based lateral flow assay (Gao et al. Biosens & Bioelectro, 2014, 54, 578–584). The method was applied to detect miR-21 in cancer cells and spiked human serum with satisfactory results.

High-Resolution Vertical Polarization Excited Dark-Field Microscopic Imaging of Anisotropic Gold Nanorods for the Sensitive Detection and Spatial Imaging of Intracellular microRNA-21.

As an important biomarker for early diagnosis of cancers, sensitive detection and high-resolution imaging of microRNA-21 in cancer cells have become important and challengeable. In this work, highly sensitive detection and spatial imaging of intracellular microRNA-21 were realized by the reduced signal background through vertical polarization excitation with a polarizer. The lateral local surface plasmon resonance property of gold nanorods (AuNRs) displayed a pronounced green color with low scattering intensity, which was adjusted to red color with strong scattering intensity when the core-satellite gold nanoparticle (AuNP) assembly was constructed on the side of AuNRs through a catalyzed hairpin assembly (CHA) circuit in the presence of microRNA-21. This unique approach allows for effectively reducing the strong background signal to improve the sensitivity of detection. Additionally, the proposed strategy can not only realize the sensitive detection of microRNA-21 with the limit of detection as low as 2 pM (3σ) but also achieve the high spatial imaging of cancer cells, which provided a specific strategy for the construction and imaging of intracellular imaging probes. It is believed that the simple and sensitive approach on the basis of lateral local surface plasmon resonance property of anisotropic AuNRs with excellent sensitivity combined with high spatial imaging holds promising potentials to visualize intracellular microRNAs with low abundance.

Analysis and Identification of Tumorigenic Targets of MicroRNA in Cancer Cells by Photoreactive Chemical Probes.

Photoactive RNA probes have unique advantages in the identification of microRNA (miR) targets due to their ability for efficient conjugation to the target sequences by covalent crosslinking, providing stable miR-mRNA complexes for further analysis. Here, we report a highly efficient and straightforward method for miR target identification that is based on photo-reactive chemical probes and RNA-seq technology (denotes PCP-Seq). UV reactive probes were prepared by incorporating psoralen in the specific position of the seed sequence of miR. Cancer cells that were transfected with the miR probes were treated with UV, following the isolation of poly(A) RNA and sequencing of the transcriptome. Quantitative analysis of RNA-seq reads and subsequent validation by qPCR, dual luciferase assay as well as western blotting confirmed that PCP-Seq could highly efficiently identify multiple targets of different miRs in the lung cancer cell line, such as targets PTTG1 and PTGR1 of miR-29a and ILF2 of miR-34a. Collectively, our data showed that PCP-Seq is a robust strategy for miR targets identification, and unique in the identification of the targets that escape degradation by miRISC and maintain normal cellular level, although their translation is repressed.

A Janus 3D DNA nanomachine for simultaneous and sensitive fluorescence detection and imaging of dual microRNAs in cancer cells.

Herein, a Janus three-dimensional (3D) DNA nanomachine was constructed for the simultaneous and sensitive fluorescence detection and imaging of dual microRNAs (miRNAs) in cancer cells, which could effectively eliminate signal interference in a homogeneous nanoparticle-based 3D DNA nanostructure caused by the proximity of the two different signal probes to achieve accurate co-location in the same position of living cancer cells. In this system, the Janus nanoparticles were synthesized as the carrier for immobilizing two different oligonucleotides on two different functionalized hemispheres of the nanoparticles to form a Janus 3D DNA nanostructure, which could convert trace amounts of miRNA-21 and miRNA-155 targets into massive FAM and Cy5-labeled duplexes to induce two remarkable fluorescence emissions by the catalytic hairpin assembly (CHA) and 3D DNA walker cascade nucleic acid amplification strategy, realizing sensitive detection and imaging of miRNA targets in cancer cells. Impressively, in comparison with current miRNA imaging methods based on nanoparticle assemblies, the proposed strategy could efficiently eliminate “false positive” results obtained in single type miRNA detection and distinctly increase the immobilization concentration of two different signal probes using Janus nanoparticles as the carrier to further enhance fluorescence intensity, resulting in accurate co-location in the same position of living cells. Meanwhile, the proposed fluorescence imaging technology makes it possible to visualize low concentrations of miRNAs with tiny change associated with some cancers, which could significantly improve the accuracy and precision compared to those of the conventional fluorescence in situ hybridization (FISH) approach. Therefore, it could serve as persuasive evidence for supplying accurate information to better understand biological processes and investigate mechanisms of various biomolecules and subcellular organelles, resulting in the further validation of their function in tumor proliferation and differentiation. This strategy provided an innovative approach to design new generations of nanomachines with ultimate applications in bioanalysis and clinical diagnoses.

Biodegradable MnO2 nanosheet based DNAzyme-recycling amplification towards: Sensitive detection of intracellular MicroRNAs

A new class of intracellular signal amplification approach, integrating biodegradable manganese dioxide (MnO2) nanosheet with target-triggered DNAzyme recycling amplification in one nanosystem, was developed for highly specific and sensitive monitoring of microRNAs (miRNAs) in living cells. Briefly, the MnO2 nanosheets were employed as carrier and quencher for the hairpin-locked-DNAzyme strands (H1). Upon entering cells, the surface-adsorbed strands (H1) can be released due to the degradation of the MnO2 nanosheets by cellular glutathione. Subsequently, the hybridization reaction between target miRNAs and H1 probe induced the conformation alteration of the hairpin probes H1, formed an “active” DNAzyme. With the assistor of Mg2+, the DNAzyme was activated and induced the release of the fluorophores labeled DNA fragment, which achieved the restoration of fluorescence signal. Meanwhile, the target molecules was released and hybridized with the other H1 strand to initiate another cycle of activation, cleavage, and turnovers, which producing enhanced fluorescence signal for sensitive analysis of intracellular miRNAs. Furthermore, fluorescence imaging experiments demonstrated that the MnO2-DNAzyme nanosystem could visually detect microRNA-21 in cancer cells. The proposed strategy provides a good platform for highly sensitive detection and imaging analysis of various intracellular miRNAs in situ.

A tRNA fragment, 5'-tiRNAVal, suppresses the Wnt/β-catenin signaling pathway by targeting FZD3 in breast cancer.

tRNA-derived fragments offer a recently identified group of non-coding single-stranded RNAs that are often as abundant as microRNAs in cancer cells and play important roles in carcinogenesis. However, the biological functions of them in breast cancer are still unclear. Hence, we focused on investigating whether tiRNAs could play a key role in the progression of breast cancer. We have identified 5'-tiRNAVal with significantly low expression in breast cancer tissues. The down-regulation of serum 5'-tiRNAVal was positively correlated with stage progression and lymph node metastasis. Overexpression of 5'-tiRNAVal suppressed cells malignant activities. FZD3 was confirmed to be a direct target of 5'-tiRNAVal in breast cancer. In addition, FZD3, β-Catenin, c-myc and cyclinD1 levels in 5'-tiRNAVal overexpressing cells were downregulated while APC was inversely upregulated. Moreover, 5'-tiRNAVal inhibited the FZD3-mediated Wnt/β-Catenin signaling pathway in breast cancer cells. Finally, 5'-tiRNAVal levels differentiated breast cancer from healthy controls with a sensitivity of 90.0% and specificity of 62.7%. This is the first study to show that 5'-tiRNAVal as a new tumor-suppressor through inhibition of FZD3/Wnt/β-Catenin signaling pathway, which could be as a potential diagnostic biomarker for breast cancer.