LncRNA PART1 promotes malignant biological behaviours associated with head and neck cancer cells via synergistic action with FUT6.

The aim of this study was to determine the role of lncRNA PART1 and downstream FUT6 in tumorigenesis and progression of head and neck cancer (HNC). Bioinformatics analysis and qRT-PCR revealed that lncRNA PART1 was expressed at low levels in HNC patients. The proliferation, apoptosis, migration and flow cytometry results showed that low expression of lncRNA PART1 inhibited apoptosis and promoted HNC cell migration and proliferation. In addition, animal experiments have also shown that low expression of lncRNA PART1 can promote tumor growth. LncRNA PART1 overexpression promoted apoptosis and inhibited HNC cell migration and proliferation. Through bioinformatics analysis, FUT6 was found to be expressed at low levels in HNC and to be correlated with patient survival. Immunohistochemical and qRT-PCR results revealed that FUT6 was underexpressed in tumour tissues and HNC cells. Cell and animal experiments showed that overexpression of FUT6 could inhibit tumour proliferation and migration. Bioinformatics analysis revealed that lncRNA PART1 was positively correlated with FUT6. By qRT-PCR and western blot, we observed that after knockdown of lncRNA PART1, both the mRNA and protein expression levels of FUT6 were reduced. The above results indicated that lncRNA PART1 and FUT6 play an important role in HNC, and that lncRNA PART1 affected the development of tumor by downstream FUT6.

Self-Priming Cyclic Amplification Accelerating CRISPR Sensor for Sensitive and Specific MicroRNA Analysis with No Background.

In this work, we demonstrate for the first time the application of the phosphorothioated-terminal hairpin formation and self-priming extension (PS-THSP) reaction for miRNA assays. A self-priming amplification accelerating CRISPR sensor was well-established for sensitive and specific miRNA detection by integrating the PS-THSP reaction and CRISPR/Cas12a system. The sensor consists of three steps: (1) the formation of a complete PS-THSP template in the presence of target miRNA and ligase; (2) the exponential isothermal amplification of the PS-THSP reaction under the action of DNA polymerase; (3) the activation of the CRISPR/Cas12a fluorescence system to generate signals. We used miR-21 as a model target. The sensor can achieve sensitive detection of miR-21 without the involvement of any primers, and the special design of the CRISPR proto-spacer neighbor motif (PAM) sequence effectively avoids the interference of the background signal. In addition, the sensor can not only identify single-base mutant homologous sequences but also show stable performance in complex biological matrices. We have successfully used this sensor to accurately analyze miR-21 in different cell lines and real clinical samples, demonstrating its great potential in clinical diagnosis.

Asymmetrically split DNAzyme-based colorimetric and electrochemical dual-modal biosensor for detection of breast cancer exosomal surface proteins.

Exosomal surface proteins are potentially useful for breast cancer diagnosis and awareness of risk. However, some detection techniques involving complex operations and expensive instrumentation are limited to advance to clinical applications. To solve this problem, we develop a dual-modal sensor combining naked-eye detection and electrochemical assay of exosomal surface proteins from breast cancer. Most of existing sensors rely on aptamers recognizing exosomes and generating amplified signals at the same time, which require well-designed aptamer probes to avoid difficulties in identifying exosomes. In our work, aptamers not bound by the exosomes can serve as complete templates to induce formation of G quadruplexes. The peroxidase activity of the G-quadruplex/hemin DNAzyme catalyze substrates can generate both color and electrochemical signals. The developed dual-modal sensor offers a remarkable capability to differentiate nonmetastatic, metastatic breast cancer patients, and healthy individuals through the analysis of exosomal surface proteins. The sensor's distinctive features, including its universality, simplicity, and cost-effectiveness, position it as a promising diagnostic tool in breast cancer research and clinical practice.

Triple Amplification Strategy of CHA-PER-DNAzyme for Ultrasensitive Detection of circRNA Associated with Hepatocellular Carcinoma.

Biological and clinical studies have indicated that aberrant expression of circMTO1 served as a crucial biomarker for the diagnosis and prognosis of hepatocellular carcinoma (HCC) patients as well as a potential therapeutic target. However, the detection of circRNAs currently faces challenges such as homologous linear RNA interference and low-expression abundance of certain circRNAs. Therefore, we developed a triple amplification method based on catalytic hairpin assembly (CHA) activation by back-splice junction (BSJ), resulting in CHA products that triggered primer exchange reaction to generate DNAzyme. Subsequently, DNAzyme cleaved the fluorescent reporter chain, enabling ultrasensitive detection of hepatocellular carcinoma-associated circMTO1 through the output fluorescence signal. The catalytic hairpin opening sequence within CHA specifically targeted the BSJ sequence in circRNA, thereby avoiding false positive signals observed in circRNA assays due to the recognition of homologous linear RNA molecules. Moreover, this triple amplification method facilitated sensitive detection of circRNA and addressed the issue of low-abundance expression levels associated with circMTO1 in HCC samples. Notably, our newly designed assay for detecting circRNA exhibited a linear range from 1 fM to 100 nM with a detection limit of 0.265 fM. Furthermore, it demonstrated excellent and consistent performance even within complex systems. Our proposed method enabled the specific and sensitive detection of circMTO1 in various cancer cells and blood samples from HCC patients, providing an innovative approach for investigating the role of circRNA in tumorigenesis and development while promoting its clinical application.

Accelerating Cell-Free Biosensors by Entropy-Driven Assembly of Transcription Templates.

Due to its high efficiency and selectivity, cell-free biosynthesis has found broad utility in the fields of bioproduction, environment monitoring, and disease diagnostics. However, the practical application is limited by its low productivity. Here, we introduce the entropy-driven assembly of transcription templates as dynamic amplifying modules to accelerate the cell-free transcription process. The catalytic DNA circuit with high sensitivity and enzyme-free format contributes to the production of large amounts of transcription templates, drastically accelerating the as-designed cell-free transcription system without interference from multiple enzymes. The proposed approach was successfully applied to the ultrasensitive detection of SARS-CoV-2, improving the sensitivity by 3 orders of magnitude. Thanks to the high programmability and diverse light-up RNA pairs, the method can be adapted to multiplexing detection, successfully demonstrated by the analysis of two different sites of the SARS-CoV-2 gene in parallel. Further, the flexibility of the entropy-driven circuit enables a dynamic responding range by tuning the circuit layers, which is beneficial for responding to targets with different concentration ranges. The strategy was also applied to the analysis of clinical samples, providing an alternative for sensitively detecting the current SARS-CoV-2 RNA that quickly mutates.

Long Non-Coding RNA Detection Based on Multi-Probe-Induced Rolling Circle Amplification for Hepatocellular Carcinoma Early Diagnosis.

Long non-coding RNAs (lncRNAs) played vital roles in physiological and pathological conditions. Consistent results from cell experiments, animal experiments, and clinical studies suggested that lncRNA HULC was an oncogenic lncRNA serving as a potential diagnostic and prognostic marker of hepatocellular carcinoma. In this study, we developed a fluorescent biosensor for lncRNA HULC detection based on rolling circle amplification (RCA) induced by multi-primer probes. Multiple primer probes can not only combine with lncRNA to break its secondary structure, which was conducive to lncRNA captured by Y-shaped probes, but also trigger multiple RCA reactions to achieve signal amplification and the goal of sensitive detection of lncRNA. Compared to previous detection methods, in this scheme, we took advantage of the long sequence characteristics of lncRNA to make it a carrier that can bind multiple primers to initiate RCA. This newly designed biosensor provided a linear range from 1 pM to 100 nM with a detection limit of 0.06 pM. This method can provide a new idea for the application of isothermal amplification in detecting lncRNA. Furthermore, the application of the biosensor in liver cancer cell lines and whole blood samples from hepatocellular carcinomatosis patients also confirmed that the method had good selectivity and sensitivity to lncRNA HULC. This method offered a new way for transforming specific lncRNA into clinical application for diagnosis, prognosis, or predicting treatment response.

Highly Sensitive Aptasensor for Detecting Cancerous Exosomes Based on Clover-like Gold Nanoclusters.

Exosome-based liquid biopsy technologies play an increasingly prominent role in tumor diagnosis. However, the simple and sensitive method for counting exosomes still faces considerable challenges. In this work, the CD63 aptamer-modified DNA tetrahedrons on the gold electrode were used as recognition elements for the specific capture of exosomes. Partially complementary DNA probes act as bridges linking trapped exosomes and three AuNP-DNA signal probes. This clover-like structure can tackle the recognition and sensitivity issues arising from the undesired AuNP aggregation event. When cancerous exosomes are present in the system, the high accumulation of methylene blue molecules from DNA-AuNP nanocomposites on the surface of the electrode leads to an intense current signal. According to the results, the aptasensor responds to MCF-7 cell-derived exosomes in the concentration range from 1.0 × 103 to 1.0 × 108 particles·µL-1, with the detection limit of 158 particles·µL-1. Furthermore, the aptasensor has been extended to serum samples from breast cancer patients and exhibited excellent specificity. To sum it up, the aptasensor is sensitive, straightforward, less expensive, and fully capable of receiving widespread application in clinics for tumor monitoring.

A functional RNA/DNA circuit for one-pot detection of SARS-CoV-2 RNA.

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a functional RNA/DNA circuit. By ingeniously integrating the nucleic acid circuit technology and CRISPR/cas12a system, this method can achieve femtomolar detection of the target RNA in one step and successfully distinguish COVID-19 positive cases from clinical samples, proving its great potential for clinical application.

Universal and Flexible Signal Transduction Module Based on Overload Triggering Probe Escape for Sensitive Detection of Tau Protein.

Aptamer-based methods have attracted increasing interest due to flexible engineering, but their generality is limited by the heterogeneity of signal transduction mechanisms. Given the fact that nonlinear and large molecules are more likely to make the nanosurface overloaded, we investigated a novel signal transduction process to extend the application of aptasensors. In this work, an aptamer complementary element (ACE) is designed with a primer region to serve as the signal probe, which can fully hybridize with an aptamer and be separated by magnetic beads (MBs). Upon target binding, the formed aptamer/target complex is much larger than the linear aptamer/ACE-primer dimer, causing overload of MBs on account of steric hindrance. An extra aptamer/ACE-primer can escape from the surface to the supernatant, which can be amplified by a catalytic hairpin assembly (CHA) circle. The size-dependent signal transduction and the modular design endow the method with high generality and flexibility for protein analysis. The proposed aptasensor was successfully applied to the detection of tau proteins ranging from 0.5 to 1000 ng mL-1 with a limit of detection (LOD) as low as 0.254 ng mL-1. The recovery tests in both human serum and cerebra spinal fluid confirmed the high accuracy and stability. Furthermore, a successful distinction was made between AD patients and healthy controls by the method, suggesting the possible applicability for practical analysis of tau proteins.

Highly Sensitive Detection of the Immune Checkpoint PD-L1-Positive Circulating Tumor Cells Based on Steric Hindrance.

Programmed-death ligand 1 (PD-L1), as one of major immune checkpoints, is highly expressed on cancer cells and participates in the immune escape process of tumor cells. The level of PD-L1 in patients is closely related to the efficacy of anti-PD-L1 immunotherapy, and patients with a high level have better response to immunotherapy. Therefore, PD-L1 can be an indicator of patient classification and medication guidance. In this work, we have developed a novel strategy for detecting PD-L1-positive circulating tumor cells based on steric hindrance generated after cell capture, using the primer exchange reaction (PER) amplification method. The principle is to modify a single strand containing the PD-L1 aptamer and the PER primer on the electrode surface. When PD-L1-positive circulating tumor cells exist, the aptamer will capture them. The steric hindrance generated by the captured cells due to their large volume hinders the subsequent approach of PER materials, thus hindering the occurrence of PER signal amplification. The number of HRP bound to the electrode surface is reduced, and the current signal output is inversely proportional to the number of captured cells. This method realizes convenient and sensitive detection of PD-L1-positive tumor cells and provides a new means for clinical judgment of whether patients should adopt immunotherapy.

A simple and rapid method to assay SARS-CoV-2 RNA based on a primer exchange reaction.

A simple method is proposed in this work for the detection of SARS-CoV-2 RNA based on a primer exchange reaction (PER). By ingeniously integrating the PER cascade and CRISPR/cas12a system, this method can achieve convenient detection of the target RNA in 40 min and distinguish a single-base mutation from the target sequence, demonstrating its superior analytical performance.

Exocyst controls exosome biogenesis via Rab11a.

Tumor cells actively release large quantities of exosomes, which pivotally participate in the regulation of cancer biology, including head and neck cancer (HNC). Exosome biogenesis and release are complex and elaborate processes that are considered to be similar to the process of exocyst-mediated vesicle delivery. By analyzing the expression of exocyst subunits and their role in patients with HNC, we aimed to identify exocyst and its functions in exosome biogenesis and investigate the molecular mechanisms underlying the regulation of exosome transport in HNC cells. We observed that exocysts were highly expressed in HNC cells and could promote exosome secretion in these cells. In addition, downregulation of exocyst expression inhibited HN4 cell proliferation by reducing exosome secretion. Interestingly, immunofluorescence and electron microscopy revealed the accumulation of multivesicular bodies (MVBs) after the knockdown of exocyst. Autophagy, the major pathway of exosome degradation, is not activated by this intracellular accumulation of MVBs, but these MVBs are consumed when autophagy is activated under the condition of cell starvation. Rab11a, a small GTPase that is involved in MVB fusion, also interacted with the exocyst. These findings suggest that the exocyst can regulate exosome biogenesis and participate in the malignant behavior of tumor cells.

Orai-vascular endothelial-cadherin signaling complex regulates high-glucose exposure-induced increased permeability of mouse aortic endothelial cells.

INTRODUCTION: Diabetes-associated endothelial barrier function impairment might be linked to disturbances in Ca2+ homeostasis. To study the role and molecular mechanism of Orais-vascular endothelial (VE)-cadherin signaling complex and its downstream signaling pathway in diabetic endothelial injury using mouse aortic endothelial cells (MAECs). RESEARCH DESIGN AND METHODS: The activity of store-operated Ca2+ entry (SOCE) was detected by calcium imaging after 7 days of high-glucose (HG) or normal-glucose (NG) exposure, the expression levels of Orais after HG treatment was detected by western blot analysis. The effect of HG exposure on the expression of phosphorylated (p)-VE-cadherin and VE-cadherin on cell membrane was observed by immunofluorescence assay. HG-induced transendothelial electrical resistance was examined in vitro after MAECs were cultured in HG medium. FD-20 permeability was tested in monolayer aortic endothelial cells through transwell permeability assay. The interactions between Orais and VE-cadherin were detected by co-immunoprecipitation and immunofluorescence technologies. Immunohistochemical experiment was used to detect the expression changes of Orais, VE-cadherin and p-VE-cadherin in aortic endothelium of mice with diabetes. RESULTS: (1) The expression levels of Orais and activity of SOCE were significantly increased in MAECs cultured in HG for 7 days. (2) In MAECs cultured in HG for 7 days, the ratio of p-VE-cadherin to VE-cadherin expressed on the cell membrane and the FD-20 permeability in monolayer endothelial cells increased, indicating that intercellular permeability increased. (3) Orais and VE-cadherin can interact and enhance the interaction ratio through HG stimulation. (4) In MAECs cultured with HG, the SOCE activator ATP enhanced the expression level of p-VE-cadherin, and the SOCE inhibitor BTP2 decreased the expression level of p-VE-cadherin. (5) Significantly increased expression of p-VE-cadherin and Orais in the aortic endothelium of mice with diabetes. CONCLUSION: HG exposure stimulated increased expression of Orais in endothelial cells, and increased VE-cadherin phosphorylation through Orais-VE-cadherin complex and a series of downstream signaling pathways, resulting in disruption of endothelial cell junctions and initiation of atherosclerosis.

Orai-IGFBP3 signaling complex regulates high-glucose exposure-induced increased proliferation, permeability, and migration of human coronary artery endothelial cells.

INTRODUCTION: Diabetes-associated endothelium dysfunction might be linked to disturbances in Ca2+ homeostasis. Our main objective is to reveal the potential mechanisms by which high-glucose (HG) exposure promotes increased proliferation of human coronary artery endothelial cells (HCAECs) in culture, and that store-operated Ca2+ entry (SOCE) and insulin-like growth factor binding protein 3 (IGFBP3) contribute to this proliferation. RESEARCH DESIGN AND METHODS: We detected the expression levels of Ca2+ release-activated calcium channel proteins (Orais), IGFBP3 and proliferating cell nuclear antigen of HCAECs cultured in HG medium for 1, 3, 7, and 14 days and in streptozotocin-induced diabetic mouse coronary endothelial cells. Coimmunoprecipitation and immunofluorescence technologies were used to detect the interactions between Orais and IGFBP3 of HCAECs exposed to HG environment, and to detect IGFBP3 expression and proliferation after treatment of HCAECs cultured in HG medium with an agonist or inhibitor of SOCE. Similarly, after transfection of specific small interfering RNA to knock down IGFBP3 protein expression, SOCE activity and Orais expression were tested. Some processes related to endothelial dysfunction, such as migration, barrier function and adhesion marker expression, are also measured. RESULTS: HG exposure promoted increased proliferation of HCAECs in culture and that SOCE and IGFBP3 contributed to this proliferation. In addition, we also found that Orais and IGFBP3 were physically associated and regulated each other's expression levels. Besides, their expression levels and interactions were enhanced in HCAECs after exposure to HG. HG exposure promotes cell migration, but reduces barrier function and adherens junction protein expression levels in HCAECs. CONCLUSION: Orais and IGFBP3 formed a signaling complex that mediated HCAEC proliferation during HG exposure in culture. Meanwhile, we also found that SOCE stimulates proliferation of HCAECs by regulating IGFBP3, thereby promoting the occurrence and progression of coronary atherosclerosis in diabetes. It is worth noting that our findings may shed new light on the mechanisms of increased proliferation in HCAECs in diabetes and suggest the potential value of SOCE and IGFBP3 as therapeutic targets for coronary atherosclerosis in individuals with diabetes.

Role of TRPP2 in mouse airway smooth muscle tension and respiration.

The transient receptor potential polycystin-2 (TRPP2) is encoded by the Pkd2 gene, and mutation of this gene can cause autosomal dominant polycystic kidney disease (ADPKD). Some patients with ADPKD experience extrarenal manifestations, including radiologic and clinical bronchiectasis. We hypothesized that TRPP2 may regulate airway smooth muscle (ASM) tension. Thus, we used smooth muscle-Pkd2 conditional knockout (Pkd2SM-CKO) mice to investigate whether TRPP2 regulated ASM tension and whether TRPP2 deficiency contributed to bronchiectasis associated with ADPKD. Compared with wild-type mice, Pkd2SM-CKO mice breathed more shallowly and faster, and their cross-sectional area ratio of bronchi to accompanying pulmonary arteries was higher, suggesting that TRPP2 may regulate ASM tension and contribute to the occurrence of bronchiectasis in ADPKD. In a bioassay examining isolated tracheal ring tension, no significant difference was found for high-potassium-induced depolarization of the ASM between the two groups, indicating that TRPP2 does not regulate depolarization-induced ASM contraction. By contrast, carbachol-induced contraction of the ASM derived from Pkd2SM-CKO mice was significantly reduced compared with that in wild-type mice. In addition, relaxation of the carbachol-precontracted ASM by isoprenaline, a ß-adrenergic receptor agonist that acts through the cAMP/adenylyl cyclase pathway, was also significantly attenuated in Pkd2SM-CKO mice compared with that in wild-type mice. Thus, TRPP2 deficiency suppressed both contraction and relaxation of the ASM. These results provide a potential target for regulating ASM tension and for developing therapeutic alternatives for some ADPKD complications of the respiratory system or for independent respiratory disease, especially bronchiectasis.