SelK promotes glioblastoma cell proliferation by inhibiting ß-TrCP1 mediated ubiquitin-dependent degradation of CDK4.

BACKGROUND: Glioblastoma (GB) is recognized as one of the most aggressive brain tumors, with a median survival of 14.6 months. However, there are still some patients whose survival time was greater than 3 years, and the biological reasons behind this clinical phenomenon arouse our research interests. By conducting proteomic analysis on tumor tissues obtained from GB patients who survived over 3 years compared to those who survived less than 1 year, we identified a significant upregulation of SelK in patients with shorter survival times. Therefore, we hypothesized that SelK may be an important indicator related to the occurrence and progression of GBM. METHODS: Proteomics and immunohistochemistry from GB patients were analyzed to investigate the correlation between SelK and clinical prognosis. Cellular phenotypes were evaluated by cell cycle analysis, cell viability assays, and xenograft models. Immunoblots and co-immunoprecipitation were conducted to verify SelK-mediated ubiquitin-dependent degradation of CDK4. RESULTS: SelK was found to be significantly upregulated in GB samples from short-term survivors (≤ 1 year) compared to those from long-term survivors (≥ 3 years), and its expression levels were negatively correlated with clinical prognosis. Knocking down of SelK expression reduced GB cell viability, induced G0/G1 phase arrest, and impaired the growth of transplanted glioma cells in nude mice. Down-regulation of SelK-induced ER stress leads to a reduction in the expression of SKP2 and an up-regulation of ß-TrCP1 expression. Up-regulation of ß-TrCP1, thereby accelerating the ubiquitin-dependent degradation of CDK4 and ultimately inhibiting the malignant proliferation of the GB cells. CONCLUSION: This study discovered a significant increase in SelK expression in GB patients with poor prognosis, revealing a negative correlation between SelK expression and patient outcomes. Further mechanistic investigations revealed that SelK enhances the proliferation of GB cells by targeting the endoplasmic reticulum stress/SKP2/ß-TrCP1/CDK4 axis.

Discovery of the 2,4-disubstituted quinazoline derivative as a novel neddylation inhibitor for tumor therapy.

Overactivation of neddylation has been found in a number of common human tumor-related diseases. In recent years, targeting the neddylation pathway has become an appealing anti-cancer strategy, and it is critical to find neddylation inhibitors with novel structures and higher efficacy. Here, we present the discovery of novel inhibitors of the NEDD8-activating enzyme (NAE) and their antitumor activity in vitro. All synthesized 1,4-disubstituted piperidine compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549, and KYSE-30 cells. Among five representative compounds, III-26 bearing a quinazoline motif was identified as the lead one due to the fact that it significantly hindered the neddylation of Cullin1. Cellular mechanisms elucidated that III-26 inhibited the proliferation, migration, and invasion of UBC12-overexpressed MGC-803 cell lines, as well as induced apoptosis and arrested the cell cycle at G2/M phase. Importantly, III-26 reduced NAE activity, thus selectively preventing neddylation of Cullin3 and Cullin1 over other Cullin members. At a dose of 4 µM, III-26 virtually entirely blocked UBC12-NEDD8 conjugation in MGC-803 cells. Our molecular modeling and kinetic investigation suggested that this compound may function as a non-covalent inhibitor of NAE.

Identification of novel benzothiazole derivatives as inhibitors of NEDDylation pathway to inhibit the progression of gastric cancer.

The overexpression of neddylation modification is frequently observed in human tumor cells. Targeting the neddylation pathway has been recognized as a promising anticancer therapeutic strategy, thus discovering potent and selective neddylation inhibitors is of great importance. In this study, we designed and synthesized a series of novel neddylation inhibitors bearing benzothiazole scaffolds by molecular hybridization strategy and all compounds were evaluated for antiproliferative activity against MGC-803, MCF-7, A549 and KYSE-30 cell lines. In vitro anti-tumor studies showed that the most promising compound X-10, effectively suppressed MGC-803 cells growth and migration, induced apoptosis and arrested cell cycle at G2/M phase. Importantly, by directly interacting with NAE1, X-10 blocked NAE1 activity, specifically preventing neddylation of Cullin 3 and Cullin 1, and produced a dose-response decline in the level of UBC12-NEDD8 complex. Overall, our data indicate that X-10 inhibits the process of neddylation, making it a potentially agent for both cancer prevention and therapy purposes.

CRTAC1 enhances the chemosensitivity of non-small cell lung cancer to cisplatin by eliciting RyR-mediated calcium release and inhibiting Akt1 expression.

Sensitivity to platinum-based combination chemotherapy is associated with a favorable prognosis in patients with non-small cell lung cancer (NSCLC). Here, our results obtained from analyses of the Gene Expression Omnibus database of NSCLC patients showed that cartilage acidic protein 1 (CRTAC1) plays a role in the response to platinum-based chemotherapy. Overexpression of CRTAC1 increased sensitivity to cisplatin in vitro, whereas knockdown of CRTAC1 decreased chemosensitivity of NSCLC cells. In vivo mouse experiments showed that CRTAC1 overexpression increased the antitumor effects of cisplatin. CRTAC1 overexpression promoted NFAT transcriptional activation by increasing intracellular Ca2+ levels, thereby inducing its regulated STUB1 mRNA transcription and protein expression, accelerating Akt1 protein degradation and, in turn, enhancing cisplatin-induced apoptosis. Taken together, the present results indicate that CRTAC1 overexpression increases the chemosensitivity of NSCLC to cisplatin treatment by inducing Ca2+-dependent Akt1 degradation and apoptosis, suggesting the potential of CRTAC1 as a biomarker for predicting cisplatin chemosensitivity. Our results further reveal that modulating the expression of CRTAC1 could be a new strategy for increasing the efficacy of cisplatin in chemotherapy of NSCLC patients.

Nomograms and scoring system for forecasting overall and cancer-specific survival of patients with prostate cancer.

BACKGROUND: Estimated life expectancy is one of the most important factors in determining treatment options for prostate cancer (PCa) patients. However, clinicians have few effective prognostic tools to individually assess survival in patients with PCa. METHODS: We screened 283,252 patients diagnosed with PCa from the Surveillance, Epidemiology, and End Results (SEER) database between 2004 and 2015, and randomly divided them into the training and validation groups. We used univariate and multivariate Cox analyses to identify independent prognostic factors and further established nomograms to predict 1-, 3-, 5-, and 10-year overall survival (OS) and cancer-specific survival (CSS) for PCa patients. The prediction performance of nomograms was tested and externally validated by Concordance index (C-index) and receiver operating characteristic (ROC) curve. Calibration curve and decision curve analysis (DCA) were used for internal validation. We further developed PCa prognostic scoring system based on the impact of available variables on survival. RESULTS: The variables age, race, marital status, TNM stage, surgery method, radiotherapy, chemotherapy, PSA value, and Gleason score identified as independent prognostic factors were included in the survival nomograms. The results of training (C-index: OS = 0.776, CSS = 0.889; AUC value: OS = 0.772-0.802, CSS = 0.892-0.936) and external validation (C-index: OS = 0.759, CSS = 0.875) indicated our nomograms had good performance in predicting 1-, 3-, 5-, and 10-year OS and CSS prediction. Internal validation using the calibration curves and DCA curves demonstrated the effectiveness of the prediction models. The prognostic scoring system was more effective than the AJCC staging system in predicting the survival of PCa patients, especially for OS. CONCLUSION: The prognostic nomograms and prognostic scoring system have favorable performance in predicting OS and CSS of PCa patients. These individualized survival prediction tools may contribute to clinical decisions.

A predictive model based on ground glass nodule features via high-resolution CT for identifying invasiveness of lung adenocarcinoma.

Objective: The morphology of ground-glass nodule (GGN) under high-resolution computed tomography (HRCT) has been suggested to indicate different histological subtypes of lung adenocarcinoma (LUAD); however, existing studies only include the limited number of GGN characteristics, which lacks a systematic model for predicting invasive LUAD. This study aimed to construct a predictive model based on GGN features under HRCT for LUAD. Methods: A total of 301 surgical LUAD patients with HRCT-confirmed GGN were enrolled, and their GGN-related features were assessed by 2 individual radiologists. The pathological diagnosis of the invasive LUAD was established by pathologic examination following surgery (including 171 invasive and 130 non-invasive LUAD patients). Results: GGN features including shorter distance from pleura, larger diameter, area and mean CT attenuation, more heterogeneous uniformity of density, irregular shape, coarse margin, not defined nodule-lung interface, spiculation, pleural indentation, air bronchogram, vacuole sign, vessel changes, lobulation were observed in invasive LUAD patients compared with non-invasive LUAD patients. After adjustment by multivariate logistic regression model, GGN diameter (OR = 1.490, 95% CI, 1.326-1.674), mean CT attenuation (OR = 1.007, 95% CI, 1.004-1.011) and heterogeneous uniformity of density (OR = 3.009, 95% CI, 1.485-6.094) were independent risk factors for invasive LUAD. In addition, a predictive model integrating these three independent GGN features was established (named as invasion of lung adenocarcinoma by GGN features (ILAG)), and receiver-operating characteristic curve illustrated that the ILAG model presented good predictive value for invasive LUAD (AUC: 0.919, 95% CI, 0.889-0.949). Conclusions: ILAG predictive model integrating GGN diameter, mean CT attenuation and heterogeneous uniformity of density via HRCT shows great potential for early estimation of LUAD invasiveness.

mTOR pathway gene mutations predict response to immune checkpoint inhibitors in multiple cancers.

BACKGROUND: mTOR pathway is known to promote cancer malignancy and influence cancer immunity but is unknown for its role in immune checkpoint inhibitors (ICI) therapy. METHODS: Using Memorial Sloan-Kettering Cancer Center dataset (MSKCC), we extracted mTOR pathway gene mutations for stepwise Cox regression in 1661 cancer patients received ICI. We associated the mutation of the gene signature resulted from the stepwise Cox regression with the 1661 patients' survival. Other 553 ICI-treated patients were collected from 6 cohorts for validation. We also performed this survival association in patients without ICI treatment from MSKCC as discovery (n = 2244) and The Cancer Genome Atlas (TCGA) as validation (n = 763). Pathway enrichment analysis were performed using transcriptome profiles from TCGA and IMvigor210 trial to investigate the potential mechanism. RESULTS: We identified 8 genes involved in mTOR pathway, including FGFR2, PIK3C3, FGFR4, FGFR1, FGF3, AKT1, mTOR, and RPTOR, resulted from stepwise Cox regression in discovery (n = 1661). In both discovery (n = 1661) and validation (n = 553), the mutation of the 8-gene signature was associated with better survival of the patients treated with ICI, which was independent of tumor mutation burden (TMB) and mainly attributed to the missense mutations. This survival association was not observed in patients without ICI therapy. Intriguingly, the mutation of the 8-gene signature was associated with increased TMB and PD1/PD-L1 expression. Immunologically, pathways involved in anti-tumor immune response were enriched in presence of this mutational signature in mTOR pathway, leading to increased infiltration of immune effector cells (e.g., CD8 + T cells, NK cells, and M1 macrophages), but decreased infiltration of immune inhibitory M2 macrophages. CONCLUSIONS: These results suggested that mTOR pathway gene mutations were predictive of better survival upon ICI treatment in multiple cancers, likely by its association with enhanced anti-tumor immunity. Larger studies are warranted to validate our findings.

Tetrahedral DNA Nanostructure with Multiple Target-Recognition Domains for Ultrasensitive Electrochemical Detection of Mucin 1.

In this work, a tetrahedral DNA nanostructure (TDN) designed with multiple biomolecular recognition domains (m-TDN) was assembled to construct an ultrasensitive electrochemical biosensor for the quantitative detection of tumor-associated mucin 1 (MUC-1) protein. This new nanostructure not only effectively increased the capture efficiency of target proteins compared to the traditional TDN with a single recognition domain but also enhanced the sensitivity of the constructed electrochemical biosensors. Once the target MUC-1 was captured by the protein aptamers, the ferrocene-marked DNA strands as electrochemical signal probes at the vertices of m-TDN would be released away from the electrode surface, causing significant reduction of the electrochemical signal, thereby enhancing significantly the detection sensitivity. As a result, this well-designed biosensor achieved ultrasensitive detection of the biomolecule at a linear range from 1 fg mL-1 to 1 ng mL-1, with the limit of detection down to 0.31 fg mL-1. This strategy provides a new approach to enhance the detection sensitivity for the diagnosis of diseases.

Diagnosis and genotype-phenotype correlation in patients with PKD1/TSC2 contiguous gene deletion syndrome.

Deletions involving the TSC2 and PKD1 genes lead to tuberous sclerosis complex (TSC) and autosomal dominant polycystic kidney disease (ADPKD), which is known as TSC2-PKD1 contiguous gene deletion syndrome (PKDTS). PKDTS leads to severe symptoms and death. There are few reported cases of PKDTS, the phenotypic descriptions are poor, and detailed statistics and descriptions of the time of onset and prognosis of PKDTS are lacking. This is the first study to report on the clinical data of PKDTS patients in China. We analyzed all cases including Chinese individuals and summarized the clinical manifestations and genetic characteristics. Our study was the first to use a combination of exome sequencing and multiplex ligation-dependent probe amplification (MLPA) to screen and diagnose PKDTS. We found that many PKDTS patients have the following: multiple renal cysts; angiofibromas (≥ 3) or fibrous cephalic plaque; subependymal nodules; seizures; intellectual disability. PKDTS develops into polycystic kidney disease from before birth to 17 years old and the time of occurrence of end-stage renal disease or dialysis was 21.62 ± 12.87 years of age, which was significantly earlier than in ADPKD caused by PKD1 mutation. Compared with non-Chinese individuals of diverse ancestry, Chinese people have significant differences in the clinical characteristics, including ungual fibromas (≥ 2), and shagreen patch. Five novel large deletions were identified in Chinese. We found no relationship between the clinical phenotype and the genotype. We combined exome sequencing with MLPA to develop a diagnostic method for PKDTS.

Programmable High-Speed and Hyper-Efficiency DNA Signal Magnifier.

Herein, a programmable dual-catalyst hairpin assembly (DCHA) for realizing the synchronous recycle of two catalysts is developed, displaying high reaction rate and outstanding conversion efficiency beyond traditional nucleic acid signal amplifications (NASA). Once catalyst I interacts with the catalyst II, the DCHA can be triggered to realize the simultaneous recycle of catalysts I and II to keep the highly concentrated intermediate product duplex I-II instead of the steadily decreased one in typical NASA, which can accomplish in about only 16 min and achieves the outstanding conversion efficiency up to 4.54 × 108 , easily conquering the main predicaments of NASA: time-consuming and low-efficiency. As a proof of the concept, the proposed DCHA as a high-speed and hyper-efficiency DNA signal magnifier is successfully applied in the rapid and ultrasensitive detection of miRNA-21 in cancer cell lysates, which exploits the new generation of universal strategy for the applications in biosensing assay, clinic diagnose, and DNA nanobiotechnology.

Whole-Exome Sequencing on Circulating Tumor Cells Explores Platinum-Drug Resistance Mutations in Advanced Non-small Cell Lung Cancer.

Circulating tumor cells (CTCs) have important applications in clinical practice on early tumor diagnosis, prognostic prediction, and treatment evaluation. Platinum-based chemotherapy is a fundamental treatment for non-small cell lung cancer (NSCLC) patients who are not suitable for targeted drug therapies. However, most patients progressed after a period of treatment. Therefore, revealing the genetic information contributing to drug resistance and tumor metastasis in CTCs is valuable for treatment adjustment. In this study, we enrolled nine NSCLC patients with platinum-based chemotherapy resistance. For each patient, 10 CTCs were isolated when progression occurred to perform single cell-level whole-exome sequencing (WES). Meanwhile the patients' paired primary-diagnosed formalin-fixed and paraffin-embedded samples and progressive biopsy specimens were also selected to perform WES. Comparisons of distinct mutation profiles between primary and progressive specimens as well as CTCs reflected different evolutionary mechanisms between CTC and lymph node metastasis, embodied in a higher proportion of mutations in CTCs shared with paired progressive lung tumor and hydrothorax specimens (4.4-33.3%) than with progressive lymphatic node samples (0.6-11.8%). Functional annotation showed that CTCs not only harbored cancer-driver gene mutations, including frequent mutations of EGFR and TP53 shared with primary and/or progressive tumors, but also particularly harbored cell cycle-regulated or stem cell-related gene mutations, including SHKBP1, NUMA1, ZNF143, MUC16, ORC1, PON1, PELP1, etc., most of which derived from primary tumor samples and played crucial roles in chemo-drug resistance and metastasis for NSCLCs. Thus, detection of genetic information in CTCs is a feasible strategy for studying drug resistance and discovering new drug targets when progressive tumor specimens were unavailable.

Co-catalytic Fc/HGQs/Fe3O4 nanocomposite mediated enzyme-free electrochemical biosensor for ultrasensitive detection of MicroRNA.

Herein, a novel co-catalytic ferrocene/hemin/G-quadruplexes/Fe3O4 nanoparticles (Fc/HGQs/Fe3O4) nanocomposite was synthesized to significantly magnify the electrochemical signal of ferrocene (Fc) using the synergistic catalysis of hemin/G-quadruplexes (HGQs) and Fe3O4 nanoparticles as hydrogen peroxide enzyme mimics for the construction of ultrasensitive electrochemical biosensors. The fabricated electrochemical biosensor can achieve ultrasensitive detection of miRNA-155 ranging from 0.1 fM to 1 nM, as well as a limit of detection of 74.8 aM. This strategy provides a new route to exploring efficient signal labels for signal amplification and provides an impetus to find novel methods for the construction of biosensors for biological detection and the early clinic diagnosis of diseases.

Oncogenic role of MIR516A in human bladder cancer was mediated by its attenuating PHLPP2 expression and BECN1-dependent autophagy.

Although MIR516A has been reported to be downregulated and act as a tumor suppressor in multiple cancers, its expression and potential contribution to human bladder cancer (BC) remain unexplored. Unexpectedly, we showed here that MIR516A was markedly upregulated in human BC tissues and cell lines, while inhibition of MIR516A expression attenuated BC cell monolayer growth in vitro and xenograft tumor growth in vivo, accompanied with increased expression of PHLPP2. Further studies showed that MIR516A was able to directly bind to the 3'-untranslated region of PHLPP2 mRNA, which was essential for its attenuating PHLPP2 expression. The knockdown of PHLPP2 expression in MIR516A-inhibited cells could reverse BC cell growth, suggesting that PHLPP2 is a MIR516A downstream mediator responsible for MIR516A oncogenic effect. PHLPP2 was able to mediate BECN1/Beclin1 stabilization indirectly, therefore promoting BECN1-dependent macroautophagy/autophagy, and inhibiting BC tumor cell growth. In addition, our results indicated that the increased autophagy by attenuating MIR516A resulted in a dramatic inhibition of xenograft tumor formation in vivo. Collectively, our results reveal that MIR516A has a novel oncogenic function in BC growth by directing binding to PHLPP2 3'-UTR and inhibiting PHLPP2 expression, in turn at least partly promoting CUL4A-mediated BECN1 protein degradation, thereby attenuating autophagy and promoting BC growth, which is a distinct function of MIR516A identified in other cancers.Abbreviation: ATG3: autophagy related 3; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG12: autophagy related 12; BAF: bafilomycin A1; BC: bladder cancer; CHX: cycloheximide; Co-IP: co-immunoprecipitation; CUL3: cullin 3; CUL4A: cullin 4A; CUL4B: cullin 4B; IF: immunofluorescence: IHC-p: immunohistochemistry-paraffin; MIR516A: microRNA 516a (microRNA 516a1 and microRNA 516a2); MS: mass spectrometry; PHLPP2: PH domain and leucine rich repeat protein phosphatase.

MiRNA-516a promotes bladder cancer metastasis by inhibiting MMP9 protein degradation via the AKT/FOXO3A/SMURF1 axis.

BACKGROUND: Metastasis is the leading cause of death in patients with bladder cancer (BC). However, current available treatments exert little effects on metastatic BC. Moreover, traditional grading and staging have only a limited ability to identify metastatic BC. Accumulating evidence indicates that the aberrant expression of microRNA is intimately associated with tumor progression. So far, many miRNAs have been identified as molecular targets for cancer diagnosis and therapy. This study focused on the role of miR-516a-5p (miR-516a) in BC. METHODS: MiR-516a expression and its downstream signaling pathway were detected using molecular cell biology and biochemistry approaches and techniques. Fresh clinical BC tissue was used to study the clinicopathological characteristics of patients with different miR-516a expression. The biological functions of miR-516a in BC were tested both in vivo and in vitro. RESULTS: A more invasive BC phenotype was significantly and positively correlated with miR-516a overexpression in BC patients. MiR-516a inhibition significantly decreased BC cell invasion and migration in vitro and in vivo. Furthermore, miR-516a attenuated the expression of PH domain leucine-rich repeat-containing protein phosphatase 2 protein and inhibited SMAD-specific E3 ubiquitin protein ligase 1 transcription by activating the AKT/Forkhead box O3 signaling pathway, which stabilized MMP9 and slowed down its proteasomal degradation, ultimately promoting BC motility and invasiveness. CONCLUSIONS: Our findings reveal the crucial function of miR-516a in promoting BC metastasis, and elucidate the molecular mechanism involved, suggesting that miR-516a may be a promising novel diagnostic and therapeutic target for BC.

Effects of Transcutaneous Electrical Acupoint Stimulation on the Postoperative Sleep Quality and Pain of Patients After Video-Assisted Thoracoscopic Surgery: A Prospective, Randomized Controlled Trial.

OBJECTIVE: Postoperative sleep disturbances have serious adverse effects on postoperative outcomes. Our paper aimed to observe the effect of using transcutaneous electrical acupoint stimulation (TEAS) on sleep quality and complications after surgery in patients undergoing selective video-assisted thoracoscopic surgery. PATIENTS AND METHODS: Eighty-five patients were divided into the TEAS group or the control group randomly. Thirty minutes of TEAS treatment was performed on TEAS group at the following time points: the first night before surgery, at the end of surgery, and before sleeping on the second and third nights after surgery. The Portable Sleep Monitor (PSM) was performed to determine the sleep quality of the two nights before the operation (Sleep preop 2 and Sleep preop 1) and the first and third night after surgery (Sleep POD 1 and Sleep POD 3). The visual analog scale (VAS) was performed to evaluate pain scores after surgery and the Athens Insomnia Scale (AIS) was used for evaluating subjective sleep quality. RESULTS: Participants in the TEAS group had a lower AIS score and higher sleep efficiency at each time point except Sleep preop 2. Participants in the TEAS group showed significantly higher proportion of each sleep stage during Sleep-preop 1, Sleep POD 1, and Sleep POD 3. Patients in the TEAS group had significantly lower VAS scores at 2, 4, and 6 h during the first 24 h after surgery. The incidence of nausea and vomiting and dizziness in the control group was statistically higher than in the TEAS group. CONCLUSION: Patients usually have sleep disturbances after video-assisted thoracoscopic surgery, such as decreased distribution of each sleep stage, lower sleep efficiency, and higher AIS score. Undergoing TEAS treatment perioperatively can improve sleep quality, and effectively promote the postoperative analgesic effect and alleviate postoperative complications.

Characterization of PtAOS1 Promoter and Three Novel Interacting Proteins Responding to Drought in Poncirus trifoliata.

Jasmonic acid (JA) plays a crucial role in various biological processes including development, signal transduction and stress response. Allene oxide synthase (AOS) catalyzing (13S)-hydroperoxyoctadecatrienoic acid (13-HPOT) to an unstable allene oxide is involved in the first step of JA biosynthesis. Here, we isolated the PtAOS1 gene and its promoter from trifoliate orange (Poncirus trifoliata). PtAOS1 contains a putative chloroplast targeting sequence in N-terminal and shows relative to pistachio (Pistacia vera) AOS. A number of stress-, light- and hormone-related cis-elements were found in the PtAOS1 promoter which may be responsible for the up-regulation of PtAOS1 under drought and JA treatments. Transient expression in tobacco (Nicotiana benthamiana) demonstrated that the P-532 (-532 to +1) fragment conferring drive activity was a core region in the PtAOS1 promoter. Using yeast one-hybrid, three novel proteins, PtDUF886, PtDUF1685 and PtRAP2.4, binding to P-532 were identified. The dual luciferase assay in tobacco illustrated that all three transcription factors could enhance PtAOS1 promoter activity. Genes PtDUF1685 and PtRAP2.4 shared an expression pattern which was induced significantly by drought stress. These findings should be available evidence for trifoliate orange responding to drought through JA modulation.

One DNA circle capture probe with multiple target recognition domains for simultaneous electrochemical detection of miRNA-21 and miRNA-155.

In this work, a novel DNA circle capture probe with multiple target recognition domains was designed to develop an electrochemical biosensor for ultrasensitive detection of microRNA-21 (miRNA-21) and miRNA-155 simultaneously. The DNA circle capture probe was anchored at the top of the tetrahedron DNA nanostructure (TDN) to simultaneously recognize miRNA-21 and miRNA-155 through multiple target recognition domains under the assistance of Helper strands, which could trigger mimetic proximity ligation assay (mPLA) for capturing the beacons ferrocene (Fc)-A1 and methylene blue (MB)-A2 to achieve multiple miRNAs detection. In this way, the local reaction concentration could be enhanced and avoid the interference of various capture probes compared with the traditional multiplexed electrochemical biosensor with the use of different capture probes, resulting in the significantly improvement of detection sensitivity. As a result, this proposed biosensor showed wide linearity ranging from 0.1 fM to 10 nM with detection limits of miRNA-21 and miRNA-155 as 18.9 aM and 39.6 aM respectively, which also could be applied in the simultaneously detection of miRNA-21 and miRNA-155 from cancer cell lysates. The present strategy paved a new path in the design of capture probes for achieving more efficient and sensitive multiple biomarkers detections and possessed the potential applications in clinical diagnostic of diseases.

Simply Constructed and Highly Efficient Classified Cargo-Discharge DNA Robot: A DNA Walking Nanomachine Platform for Ultrasensitive Multiplexed Sensing.

In this work, a classified cargo-discharge DNA robot with only two DNA strands was designed and driven by an analogous proximity ligation assay (aPLA)-based enzyme cleaving for fast walk to construct a novel electrochemical biosensor for simultaneously ultrasensitive detection of microRNA-155 (miRNA-155) and miRNA-21. Compared with traditional DNA nanomachines, the multifunctional DNA robot possessed simple structure, high self-assembling efficiency and walking efficiency. Once it interacted with target miRNAs, this DNA robot could walk fast on the electrode surface and realize the classified cargoes discharging including beacons methylene blue (MB) and ferrocene (Fc), respectively labeled in the double-stranded DNA (A1-A2) for ultrasensitive detection of multiple miRNAs simultaneously. As a result, the wide linearity ranging from 100 aM to 100 pM and low detection limits of 42.7 and 51.1 aM were obtained for miRNA-155 and miRNA-21 detection, respectively. As a proof of concept, the present strategy initiates a novel and highly efficient walking platform to realize the ultrasensitive detection of biomarkers and possesses potential applications in the clinical diagnosis of disease.

Ultrasensitive Electrochemiluminescence Biosensor for Speedy Detection of microRNA Based on a DNA Rolling Machine and Target Recycling.

Intelligent DNA walking machines have become a great hot spot in biosensing, but the walking efficiency of DNA walking machines was still limited due to the low local concentration of substance DNA and the derail of leg DNA. Herein, a Zn2+-driven DNA rolling machine was proposed to overcome the above shortages and applied as a electrochemiluminescence (ECL) biosensor for speedy ultrasensitive detection of microRNA-21. First, the original DNA rolling machine was synthesized by numbers of leg DNA modified on Au nanoparticle which matched with the high concentration of track DNA on the sensing platform and could roll efficiently through Zn2+ driving. By this way the DNA rolling machine not only increased the local concentration of leg DNA and track DNA to improve walking efficiency but also changed the motion mode from step-by-step walking to high-speed rolling, weakening the derailment of leg DNA and shortening the moving time. Second, target-induced recycling and acid dissolution could convert a finite amount of target microRNA into a large amount of Zn2+, which greatly improved the sensitivity of biosensor and overcame the drawbacks of enzyme cleavage or polymerization in common nucleic acid amplification methods. Lastly, the obtained Zn2+ was employed to drive the DNA rolling machine through specific sites recognizing and track DNA cutting to remove a quencher of ferrocene, recovering ECL emission of CdS:Mn QDs for microRNA-21 detection with a detection limit of 0.28 fM. Besides, the biosensor was successfully applied in microRNA-21 analysis from human cancer cell lysates, offered a controllable and ultrasensitive strategy for speedy detection of microRNA, and revealed a new avenue for clinical analyses.

Novel 2D-DNA-Nanoprobe-Mediated Enzyme-Free-Target-Recycling Amplification for the Ultrasensitive Electrochemical Detection of MicroRNA.

In this work, on the basis of a new 2D DNA nanoprobe (DNP) and an enzyme-free-target-recycling amplification, an electrochemical biosensor is developed for the ultrasensitive detection of microRNA-21 (miRNA-21). Herein, two ferrocene-labeled bipedal DNPs, which show small steric hindrance and strong stability, are prepared on the basis of the mechanism of the proximity-ligation assay (PLA), improving the space utilization. In the presence of the target, miRNA-21, and a hairpin DNA strand, the DNP will collapse, and then two ferrocene-labeled DNA strands and the miRNA-21 will be simultaneously released from the electrode surface through toehold-mediated strand-displacement reactions (TSDRs), leading to a decrease in the electrochemical signal and realization of enzyme-free target recycling. As a result, the one input target, miRNA-21, could release 2 N ferrocene-labeled DNA strands, achieving a dramatic decrease in the electrochemical signal. Combining DNPs and enzyme-free target recycling, this proposed biosensor showed a linear dependence with miRNA-21 concentration, ranging from 1.0 fM to 10 nM with a detection limit of 0.31 fM. In addition, it is worth mentioning that this biosensor can be regenerated through incubating with three assistant-DNA strands, realizing the reuse of raw materials. Surprisingly, the elaborated biosensor provides a novel strategy for building controllable DNA nanoprobes for the sensitive detection of various biomarkers.