EIF4A3-regulated hsa_circ_0001445 can inhibit the progression of laryngeal squamous cell carcinoma via hsa-miR-432-5p-dependent up-regulation of RGMA expression.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor in the head and neck, the 5-year relative survival rate of patients diagnosed with laryngeal cancer was estimated to be 61% from 2012 to 2018. An increasing number of studies have shown that circular RNAs (circRNAs) play a key role in the occurrence and development of cancer and may function as cancer biomarkers and new therapeutic targets. At present, the research on the relationship between circRNAs and LSCC is still in its infancy and needs further exploration. In this study, we found a circRNA (hsa_circ_0001445) associated with LSCC based on bioinformatics analysis. Quantitative real-time polymerase chain reaction (qRT-PCR) assay indicated that the expression of hsa_circ_0001445 was down-regulated in LSCC tissues and cell lines. Notably, the expression of hsa_circ_0001445 was negatively correlated with aggressive clinicopathological features and poor prognosis. Then, functional experiments found that overexpression of hsa_circ_0001445 inhibited the proliferation, migration and invasion of LSCC cells and tumor growth in vivo. Mechanistically, RNA immunoprecipitation (RIP), biotin-labeled probe pull-down, luciferase reporter assay and western blot experiments were employed and found that EIF4A3 reduced the expression of hsa_circ_0001445, and the direct binding of hsa_circ_0001445 to hsa-miR-432-5p attenuated the inhibitory effect of hsa-miR-432-5p on RGMA. In summary, our research suggests that hsa_circ_0001445 may be used as a potential prognostic biomarker and therapeutic target for LSCC.

Development of Flow Cytometric Assay for Detecting Papillary Thyroid Carcinoma Related hsa-miR-146b-5p through Toehold-Mediated Strand Displacement Reaction on Magnetic Beads.

In this work, a simple enzyme-free flow cytometric assay (termed as TSDR-based flow cytometric assay) has been developed for the detection of papillary thyroid carcinoma (PTC)-related microRNA (miRNA), hsa-miR-146b-5p with high performance through the toehold-mediated strand displacement reaction (TSDR) on magnetic beads (MBs). The complementary single-stranded DNA (ssDNA) probe of hsa-miR-146b-5p was first immobilized on the surface of MB, which can partly hybridize with the carboxy-fluorescein (FAM)-modified ssDNA, resulting in strong fluorescence emission. In the presence of hsa-miR-146b-5p, the TSDR is trigged, and the FAM-modified ssDNA is released form the MB surface due to the formation of DNA/RNA heteroduplexes on the MB surface. The fluorescence emission change of MBs can be easily read by flow cytometry and is strongly dependent on the concentration of hsa-miR-146b-5p. Under optimal conditions, the TSDR-based flow cytometric assay exhibits good specificity, a wide linear range from 5 to 5000 pM and a relatively low detection limit (LOD, 3σ) of 4.21 pM. Moreover, the practicability of the assay was demonstrated by the analysis of hsa-miR-146b-5p amounts in different PTC cells and clinical PTC tissues.

A versatile magnetic bead-based flow cytometric assay for the detection of thyroid cancer related hsa-miR-221-3p in blood and tissues.

In vitro detection of low abundance biomolecules including microRNAs (miRNAs) is essential to biological research and early clinical diagnosis. In this work, a versatile magnetic bead (MB)-based flow cytometric assay was developed for the detection of hsa-miR-221-3p, which is strongly associated with papillary thyroid carcinoma (PTC). In the presence of hsa-miR-221-3p, the complementary DNA probe attached to the surface of MBs is hybridized with the target to form DNA/RNA heteroduplexes. After the recognition of the DNA/RNA heteroduplexes by PicoGreen, the fluorescence signals of each MB were readily detected using a flow cytometer. This assay can selectively detect hsa-miR-221-3p with a detection limit of 2.1 pM. The practicality of the assay is demonstrated by the discrimination of thyroid cancer tissues from normal tissues, and a satisfactory result is obtained. Moreover, this assay can be rapidly carried out in one step at room temperature, providing a generic method for the sensitive detection of miRNAs in molecular diagnosis.

A DNA tetrahedron nanoprobe-based fluorescence resonance energy transfer sensing platform for intracellular tumor-related miRNA detection.

Accurate and sensitive detection of disease-related microRNAs (miRNAs) is of great significance for early disease diagnosis. In this work, a DNA tetrahedron nanoprobe (DTNP)-based fluorescence resonance energy transfer (FRET) sensing platform (termed DTNP sensor) was constructed for sensitive detection of tumor-related miRNA (e.g., hsa-miR-146b-5p) with DNA assisted cyclic amplification. DTNP was synthesized by DNA self-assembly. In the absence of hsa-miR-146b-5p, the fluorescence DNA (HP) modified with FAM at the 5' terminal and TAMRA at the 3' terminal cannot form the hairpin structure because of the hybridization with the extended DNA strand of the DNA tetrahedron, resulting in a low FRET effect. In the presence of hsa-miR-146b-5p, it would complementarily hybridize with the extended DNA strand of the DNA tetrahedron, leading to the release of HP and occurrence of strong FRET. Thus, the concentration of hsa-miR-146b-5p can be revealed by the change in the fluorescence intensity. Moreover, an assistant DNA was employed to replace hsa-miR-146b-5p for cyclic signal amplification, which can further enhance the detection sensitivity. Under the optimal experimental conditions, the limit of detection for hsa-miR-146b-5p was as low as 6 pM (S/N = 3). Furthermore, the DTNP sensor was successfully applied to evaluate the hsa-miR-146b-5p expression levels in different cell lines. The inhibition of hsa-miR-146b-5p expression in different cells was also investigated and a satisfactory result was obtained.

Polyacrylamide/Phytic Acid/Polydopamine Hydrogel as an Efficient Substrate for Electrochemical Enrichment of Circulating Cell-Free DNA from Blood Plasma.

A facile method has been developed for the rapid and efficient enrichment of DNAs from different media including synthetic single-strand DNAs (ssDNAs) from buffer solutions and cell-free DNAs (cfDNAs) from blood plasma through electric field-driven adsorption and desorption of DNAs by a polyacrylamide/phytic acid/polydopamine (PAAM/PA/PDA) hydrogel. The as-prepared PAAM/PA/PDA hydrogel possesses regular porosity with a large surface area, strong electric field responsiveness/good conductivity, and a rich aromatic structure, which can be used as an ideal adsorbent for DNA enrichment under a positive electric field. The enriched DNAs can be released efficiently when the positive electric field is converted to a negative electric field. The PAAM/PA/PDA hydrogel-based electrochemical method enables the completion of the process of DNA adsorption and release within 5 min and exhibits reasonable enrichment efficiencies and recovery rates of various DNAs. For instance, the high enrichment sensitivity (0.1 pmol L-1) together with the excellent recovery (>75%) of an ssDNA with 78 nucleotides is obtained. Combined with the PCR amplification technique, the practicability of the as-proposed method is demonstrated by the screening of circulating tumor DNAs (ctDNAs) with a BRAFV600E mutation in cfDNAs from the blood plasma samples of patients with papillary thyroid cancer or thyroid nodule and random patients from a clinical laboratory.

DNA microarray-based resonance light scattering assay for multiplexed detection of DNA mutation in papillary thyroid cancer.

Highly accurate analysis of single-nucleotide polymorphisms (SNPs) plays an important role in both disease diagnostics and personalized medicine development. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of papillary thyroid carcinoma (PTC) related mutation points including BRAFV600E (t1m), NRAS codon 61 (t2m), TERT promoter g.1295228 (t31m) and TERT promoter g.1295250 (t32m) with high sensitivity and selectivity by the attachment of polyvalent ssDNA modified 13 nm gold nanoparticles (ssDNAs@GNPs) followed by silver deposition for signal enhancement. The microarray-based RLS assay provides a detection limit (S/N = 3) at the sub-nanomolar level for the target ssDNAs and determines allele frequencies as low as 0.2% for t1m, 0.2% for t2m, 0.5% for t31m, and 0.5% for t32m in the cocktail of target ssDNAs, respectively. The practicability of the DNA microarray-based RLS assay is demonstrated by profiling of t2m in 50 clinical thyroid tissue samples of PTC patients, and satisfactory results are obtained.

Multiplexed detection of microRNAs by a competitive DNA microarray-based resonance light scattering assay.

The reliable detection and monitoring of microRNAs (miRNAs) are of great significance for gaining a better understanding of the functions of miRNAs in a wide range of biological processes. In this work, a competitive DNA microarray-based resonance light scattering (RLS) assay has been developed for the multiplexed detection of miRNAs with relatively high sensitivity and selectivity. After one-step competition hybridization reactions of miRNAs and multiple single strand DNA (ssDNA) conjugated gold nanoparticles (ssDNAsn@GNPs) with immobilized ssDNA probes on the dendrimer-modified slide, the captured ssDNAsn@GNPs are further enlarged through silver deposition. The silver enlarged ssDNAsn@GNPs can generate strong RLS under white light excitation. The simultaneous detection of multiple miRNAs can be easily achieved by monitoring the RLS signal changes of microarrays. In the proof of concept experiment, eight miRNA let-7 family members are detected with high specificity down to 0.2 pM, 0.8 pM, 0.2 pM, 0.2 pM, 0.2 pM, 0.8 pM, 0.8 pM, and 0.8 pM for miRNA let-7a to 7g, and 7i, respectively. Furthermore, the expression levels of eight miRNA let-7 family members in the total RNA extracts from five cell lines have been evaluated, and satisfactory results are obtained.

Poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) Brushes as Peptide/Protein Microarray Substrate for Improving Protein Binding and Functionality.

We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained.

Multiple detection of single nucleotide polymorphism by microarray-based resonance light scattering assay with enlarged gold nanoparticle probes.

The mapping of specific single nucleotide polymorphisms (SNPs) in patients' genome is a critical process for the development of personalized therapy. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of breast cancer related SNPs with high sensitivity and selectivity. After hybridization of the desired target single-stranded DNAs (ssDNAs) with the ssDNA probes on a microarray, the polyvalent ssDNA modified 13 nm gold nanoparticles (GNPs) are employed to label the hybridization reaction through the formation of a three-stranded DNA system. The H2O2-mediated enlargement of GNPs is then used to enhance the RLS signal. The microarray-based RLS assay provides a detection limit of 10 pM (S/N = 3) for the target ssDNA and determines an allele frequency as low as 1.0% in the target ssDNA cocktail. Combined with an asymmetric PCR technique, the proposed assay shows good accuracy and sensitivity in profiling 4 SNPs related to breast cancer of three selected cell lines.