MICROCHIMICA ACTA
·
2017
·Vol. 184
·期 11
·4359-4365
·
IF 4.58
Zhou Yunlei,Li Bingchen,Wang Minghui,Wang Jun,Yin Huanshun,Ai Shiyun
The authors describe a fluorometric assay for microRNA. It is based on two-step amplification involving (a) strand displacement replication and (b) rolling circle amplification. The strand displacement amplification system is making use of template DNA (containing a sequence that is complementary to microRNA-21) and nicking enzyme sites. After hybridization, the microRNA strand becomes extended by DNA polymerase chain reaction and then cleaved by the nicking enzyme. The DNA thus produced acts as a primer in rolling circle amplification. Then, the DNA probe SYBR Green II is added to bind to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA. The method has a wide detection range that covers the10 f. to 0.1 nM microRNA concentration range and has a detection limit as low as 1.0 fM. The method was successfully applied to the determination of microRNA-21 in the serum of healthy and breast cancer patients.
Schematic of a fluorometric microRNA assay based on two-step amplification involving strand displacement replication and rolling circle amplification. DNA probe SYBR Green II is then bound to ssDNA to generate a fluorescent signal which increases with increasing concentration of microRNA.
研究领域:
分子生物学
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生物技术
·
癌症研究
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DOI
高影响因子
ANALYTICAL BIOCHEMISTRY
·
2017
·
IF 2.334
Zhi Li,Bingchen Li,Yunlei Zhou,Huanshun Yin,Jun Wang,Shiyun Ai
It is extremely important for quantifying trace microRNAs in the biomedical applications. In this study, an ultrasensitive, rapid and efficient label-free fluorescence method was proposed and applied for detecting microRNA-21 in serum of gastric cancer patients based on DNA hybridization chain reaction (HCR). DNA H1 and DNA H2 were designed and used as hairpin probes, the HCR was proceeded in the presence of target microRNAs. Amounts of SYBR Green І dyes were used as signal molecules to intercalate long DNA concatemers from HCR, which guaranteed the model of label-free fluorescence and strong fluorescence density. The detection method showed a wide linear region from 1 fM to 10 5 fM, and the limit of detection was 0.2554 fM (at S/N = 3) for microRNAs. The results showed that this method had an excellent specificity and reproducibility. Furthermore, the label-free fluorescence strategy exhibited a sensitive response to microRNA-21 in real serum samples of gastric cancer patients and the results obtained were in accordance with reference method (R 2 = 0.994). Overall, the proposed strategy could be satisfactory for rapid, ultrasensitive and efficient detection of microRNA-21, and held great potentials in clinic diagnosis of gastric cancer.
研究领域:
生物技术
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医学诊断
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Xiaolun Peng,Huangli Yan,Zhen Wu,Wei Wen,Xiuhua Zhang,Shengfu Wang
Detection of biomarkers at the cellular level can provide more accurate and comprehensive information that is important for early diagnosis of diseases and evaluation of new drugs. However, the interference of a large number of components in cells and the requirement of high sensitivity bring great challenges for their detection. Herein, a robust and enzyme-free electrochemical platform was proposed for microRNA-21 (miRNA-21) detection by integrating the efficient separation of magnetic nanobeads (MBs) with the multisignal amplification of strand displacement amplification (SDA) and electrochemically mediated atom transfer radical polymerization (eATRP). The eATRP is capable of de novo growth of a number of electroactive polymers on the electrode surface for signal amplification. Compared to simple hybridization, SDA and eATRP can enhance the signals by ∼35-fold, achieving high signal-to-noise ratio for low-abundant target detection. Owing to their superparamagnetism and strong magnetic response ability, MBs endow the method with excellent specificity and anti-interference ability to detect miRNA-21 in cells. Using MBs as capture carriers, SDA and eATRP for signal amplification, and gold nanoflower (AuNF)-modified electrodes as working electrodes, as low as 0.32 aM miRNA-21 was detected. Furthermore, the successful detection of miRNA-21 in cells indicated the great prospect of this method in the early diagnosis of cancers, life science research, and single-entity electrochemical detection.
研究领域:
生物医学工程
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分子诊断学
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电化学传感器
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DOI
高影响因子
Journal of Analytical Science and Technology
·
2025
·Vol. 16
·期 1
·1-7
·
IF 2.8
Zhang Zhe,Xie Qiaoyi,Zhu Chenbo,Shao Huali,Wu Boying
Alterations in microRNA (miRNA) expression profiles play a pivotal role in the initiation and progression of various diseases, including pediatric pneumonia. Consequently, the development of sensitive, specific, and precise methodologies for miRNA detection represents a promising tool for clinical diagnosis and therapeutic intervention. This study proposes a highly sensitive miRNA detection approach utilizing catalytic hairpin assembly (CHA)-mediated probe release through chain extension and cascade signal amplification. The method enables precise discrimination of target miRNAs from other RNA species via a dual-target recognition mechanism. Furthermore, the incorporation of four signal amplification cycles confers substantial amplification efficiency, permitting accurate detection of low-abundance miRNAs. This system employs cascade isothermal amplification, demonstrating a linear response across concentrations from 1 fM to 100 pM and achieving a detection limit of 0.54 fM. The methodology exhibits significant potential for practical application in complex biological samples, providing a novel platform for profiling miRNA expression patterns and elucidating their mechanistic roles in disease pathogenesis.
研究领域:
分子诊断学
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RNA生物学
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生物医学工程
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临床检验医学
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DOI
高影响因子
Frontiers in Pharmacology
·
2024
·
IF 4.4
Du Ailin,Chen Yingying,Qiao Siyu,Dong Jiaxing,Li Yulin,Cao Bokai,Zhao Rongyu,Zhang Ruiling
Introduction: Chronic alcoholism is one of the most common neurological diseases in modern society. However, the key mechanisms underlying learning and memory impairments caused by chronic alcohol exposure remain unclear. In this study, a microRNA-messenger RNA (miRNA-mRNA) network was constructed to explore the potential function of key genes in chronic alcohol exposure, their effects on the hippocampus, and their mechanisms which facilitate brain injury in mice.Methods: The Morris water maze test was used to assess the learning ability of mice in each group. Mitochondrial ATPase activity and H2S levels in the hippocampi of mice were determined. Differentially expressed miRNAs and mRNAs in the mouse hippocampus were identified using second-generation sequencing. Using the TargetScan, miRTarBase, and miRDB databases, we predicted miRNA target genes and constructed a miRNA-mRNA regulatory network. Furthermore, using the Gene Ontology and KEGG databases we performed functional enrichment and protein-protein interaction analyses. Real-time quantitative polymerase chain reaction (qPCR) and other methods were employed to verify the mRNA expression of related genes.Results: The Morris water maze test revealed that mice exposed to chronic alcohol exhibited a significantly reduced learning ability compared to the control group (p < 0.05). Compared with the control group, the activity of mitochondrial ATPase in the hippocampal tissue of alcohol-treated mice was significantly decreased (p < 0.01), suggesting brain injury. In the model group, H2S significantly increased in the mice hippocampi (p < 0.01), indicating that chronic alcohol exposure could activate cystathionineβ-synthase (CBS) and catalyze the mass formation of H2S, suggesting brain injury. A total of 208 differentially expressed miRNAs and 377 differentially expressed mRNAs were screened through bioinformatic analysis. Enrichment analysis indicated that the main pathways were involved in neurodegeneration and regulation of the Wnt signaling pathway. The PCR detected a significant downregulation in the expressions of FOS and EGR1 genes.Discussion: Consequently, chronic alcohol exposure may regulate the expression of FOS and EGR1 in the hippocampus through miR-222-3p, miR-132-3p, miR-212-3p, and miR-191-5p, reduce the activity of hippocampal mitochondrial ATPase, activate CBS, catalyze the large amount of H2S formation, and destroy the mitochondrial structure, resulting in decreased learning ability. Our findings revealed valuable genes and miRNAs for the study of chronic alcohol exposure.
研究领域:
神经生物学
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分子生物学
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生物信息学
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产品关联
DOI
PubMed
高影响因子
MICROCHIMICA ACTA
·
2020
·Vol. 187
·期 6
·1-8
·
IF 6.232
Li Qing,Liang Xuehua,Mu Xiaomei,Tan Li,Lu Jiangnan,Hu Kun,Zhao Shulin,Tian Jianniao
Using 6-carboxyfluorescein (FAM) and tetramethyl rhodamine (TAMRA) as fluorescent signals a ratiometric fluorescent three-dimensional (3D) DNA walker based on a catalytic hairpin assembly (CHA) reaction for microRNA-122 detection was constructed. This method uses CHA reaction triggered indirectly by the target to mediate the 3D DNA walker operation to amplify the signal. The dual emission ratio fluorescent signal with a single excitation wavelength was used as the signal output. This strategy combines DNA walker with CHA reaction and proportional fluorescence signal output methods, which can effectively reduce the background fluorescence signal and the risk of generating false-positive signals. Thus, the impact of environmental factors on the experiment is reduced, thereby obtaining reliable and stable experimental results. It uses the fluorescence excitation wavelength of 488 nm and the maximum fluorescence emission wavelength of 520 nm and 580 nm, respectively. It has a good linear response at a microRNA concentration range of 156.0 pM ~ 7.00 nM and a detection limit of 42.94 pM. This strategy has been successfully applied to detect microRNAs in spiked serum samples.
Schematic representation of three-dimensional (3D) DNA walker constructed using catalytic hairpin self-assembly reaction (CHA)-assisted amplification and ratiometric fluorescence signal output for the detection of miRNA-122 closely related to hepatitis
研究领域:
生物化学
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分子生物学
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纳米技术
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分析化学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2022
·
IF 8.008
Guanyu Chen,Wenqian Chen,Lilan Xu,Huixuan Jin,Weiming Sun,Jianming Lan,Fang Wu,Xi Zhang,Jing Zhang,Jinghua Chen
It remains a huge challenge to integrate the sensitivity, stability, reproducibility, and anti-fouling ability of electrochemical biosensors for practical applications. Herein, we propose a self-assembled electrode combining hexanethiol (HT), poly-adenine (poly-A), and cholesteryl-modified DNA to meet this challenge. HT can tightly pack at the electrode interface to form a hydrophobic self-assembled monolayer (SAM), effectively improving the stability and signal-to-noise ratio (SNR) of electrochemical detection. Cholesteryl-modified DNA was immobilized at the electrode through the hydrophobic interaction with HT to avoid the competition between the SAM and the DNA probe on the gold site. Thus, the assembly efficiency and uniformity of the DNA probe as well as the detection reproducibility were increased remarkedly. Poly-A was added on the HT assembled electrode to occupy the unreacted sites of gold to further enhance the anti-fouling ability. The combination of HT and poly-A allows the electrode to ensure favorable anti-fouling ability without sacrificing the detection performance. On this basis, we proposed a dual-signal amplification electrochemical biosensor for the detection of exosomal microRNAs, which showed excellent sensitivity with a detection limit down to 1.46 aM. Importantly, this method has been successfully applied to detect exosomal microRNA-21 in cells and human serum samples, proving its potential utility in cancer diagnosis.
研究领域:
生物医学工程
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分析化学
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分子诊断
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DOI
PubMed
高影响因子
MOLECULES
·
2020
·Vol. 25
·期 5
·1022
·
IF 3.267
Yue Zhang,Yifang Xie,Leifang Zhang,Hang Zhao
Smoke inhalation causes acute lung injury (ALI), a severe clinical disease with high mortality. Accumulating evidence indicates that microRNA-155 (miR-155) and suppressor of cytokine signaling 1 (SOCS-1), as mediators of inflammatory response, are involved in the pathogenesis of ALI. In this paper, we explored the proinflammatory mechanism of miR-155 in smoke-inhalation-induced ALI. Our data revealed that smoke inhalation induces miR-155 expression, and miR-155 knockout (KO) significantly ameliorates smoke-inhalation-induced lung injury in mice. Neutrophil infiltration and myeloperoxidase (MPO), macrophage inflammatory protein 2 (MIP-2) and keratinocyte chemoattractant (KC) expressions were decreased in miR-155–/–mice after smoke inhalation as well. Real-time RT-PCR and immunoblotting results showed that SOCS-1 level was remarkably increased in miR-155–/–mice after smoke exposure. Furthermore, the experiments performed in isolated miR-155 KO pulmonary neutrophils demonstrated that the lack of SOCS-1 enhanced inflammatory cytokines (MIP-2 and KC) secretion in response to smoke stimulation. In conclusion, smoke induces increased expression of miR-155, and miR-155 is involved in inflammatory response to smoke-inhalation-induced lung injury by inhibiting the expression of SOCS-1.Keywords:acute lung injury;neutrophils;microRNA-155;suppressor of cytokine signaling-1
研究领域:
呼吸病学
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免疫学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
Experimental and Therapeutic Medicine
·
2020
·Vol. 20
·期 6
·1-1
·
IF 1.785
Zhirong Zhu,Guiliang Tang,Jiajun Yan
Prostate cancer (PCa), an epithelial malignancy that occurs in the prostate, is the second leading cause of cancer death worldwide. MicroRNAs (miRs/miRNAs) are reported to have important applications in the field of cancer diagnosis and treatment. The present study aimed to investigate the function of miRNA‑122 in the chemoresistance of PCa cells and the underlying mechanism. Significantly decreased miR‑122 and increased pyruvate kinase (PKM2) levels were observed in docetaxel‑resistant PCa cells, and PKM2 was negatively correlated with miR‑122. MiR‑122 mimic transfection in docetaxel‑resistant LNCaP cells significantly inhibited cell proliferation, promoted apoptosis and decreased glucose uptake and lactate production, which was counteracted by PKM2 overexpression. Inhibition of miR‑122 in LNCaP cells had an opposite effect to miR‑122 mimic transfection. In addition, miR‑122 mimic transfection significantly increased the sensitivity of docetaxel‑resistant LNCaP cells to docetaxel, while inhibition of miR‑122 significantly decreased the sensitivity of LNCaP cells to docetaxel. Luciferase reporter assays showed that miR‑122 regulated PKM2 expression by binding to the 3'‑untranslated region of PKM2. The results suggest that upregulation of miR‑122 could enhance docetaxel sensitivity, inhibit cell proliferation and promote apoptosis in PCa cells,possibly through the downregulation of its target protein PKM2.
研究领域:
肿瘤学
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分子生物学
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癌症研究
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产品关联
DOI
PubMed
高影响因子
JOURNAL OF COLLOID AND INTERFACE SCIENCE
·
2020
·
IF 7.489
Tianjiao Meng,Dan Zhao,Huimin Ye,Yue Feng,Huan Wang,Yufan Zhang
A hybridization chain reaction (HCR) amplification-based electrochemical impedimetric biosensor is fabricated for the quick, sensitive, and specific detection of miRNA-21 (miR-21) via monitoring of electrode interfacial property changes in real-time. Two sequences of H1 and H2 are adopted to trigger HCR amplification. A large amount of linear DNA concatemer are formed which could change the interfacial properties of the electrode. Interfacial charge transfer resistance difference ( R ct ) is probed via electrochemical impedance spectroscopy (EIS) and Randles equivalent circuit. After amplifying via HCR, oligonucleotides with negatively charged repelling [Fe(CN) 6 ] 3−/4− ions can form a spatial blockage. HCR amplification strategy markedly enhanced the electrochemical signal with a limit of detection (LOD) down to 4.63 fM (S/N = 3). This strategy exhibited excellent selectivity for three different miRNAs: miR-199a, miR-141, and miR-155. Moreover, results show that the proposed method can be applied to miR-21 detection in the total RNA extracted from five cells. This work presents an enzyme-free and label-free EIS nucleic acid sensor for sensitively and selectively detecting miR-21, offering a promising approach in early diseases diagnosis.
研究领域:
生物化学
·
分析化学
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生物技术
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分子生物学
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产品关联
DOI
PubMed
高影响因子
KAOHSIUNG JOURNAL OF MEDICAL SCIENCES
·
2020
·Vol. 37
·期 2
·111-120
·
IF 1.737
Shi Liu,Ying Gong,Xue-Dong Xu,Hui Shen,Shuai Gao,Hai-Dong Bao,Shi-Bin Guo,Xue-Feng Yu,Jian Gong
Gastric cancer is one of the most common cancers globally and has a poor prognosis. MiR-936 has been reported to regulate cell activity and tumor progression in non-small cell lung cancer, glioma, and epithelial ovarian cancer. However, the specific role and mechanism of miR-936 in gastric cancer have not been explored. In present study, gastric cancer cells were transfected with miR-936 mimic, and cell proliferation, cell cycle distribution, cell apoptosis, migration and invasion were assessed via cell-counting kit-8, flow cytometry, wound healing, and transwell assay, respectively. Dual luciferase reporter assay was used to check miR-936 binding to its downstream target. It was shown that miR-936 was downregulated in gastric cancer tissues and cells. Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) was confirmed as a direct target of miR-936 and negatively regulated its expression by miR-936. Overexpression of miR-936 suppressed cell proliferation, cell cycle progression, cell migration and invasion, and enhanced cell apoptosis in gastric cancer cells, which could be reversed by further ERBB4 overexpression. Western blot results showed that miR-936/ERBB4 axis regulated Akt-related pathways to control gastric cancer cell activities. Therefore, our data suggest that miR-936 overexpression inhibits cell proliferation and invasion and promotes cell apoptosis through Akt-related pathways by targeting ERBB4, which provides novel insight to target miR-936 or miR-936/ERBB4 axis for the treatment of gastric cancer.
研究领域:
肿瘤学
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分子生物学
·
癌症研究
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产品关联
DOI
PubMed
高影响因子
Bioengineered
·
2021
·
IF 3.269
Shanzhi Wan,Zhimin Liu,Yang Chen,Zhitao Mai,Mingming Jiang,Qingguo Di,Baohua Sun
MicroRNA (miR)-140-3p has been proved to repress lung adenocarcinoma (LUAD), and our study aims to further evaluate the mechanism. Bioinformatic analyses were performed. The viability, proliferation, migration, invasion and angiogenesis of transfected LUAD cells were all determined via Cell Counting Kit-8, colony formation, Scratch, Transwell, and tube formation assays. The targeting relationship between miR-140-3p and thymidylate synthetase (TYMS) was confirmed by dual-luciferase reporter assay. Relative expressions of miR-140-3p, TYMS, epithelial-to-mesenchymal transition- (E-cadherin, N-cadherin, vimentin), angiogenesis- (vascular endothelial growth factor (VEGF)), and apoptosis-related factors (cleaved caspase-3, B-cell lymphoma-2 (Bcl-2), Bcl-2-associated X protein (Bax)) were quantified by quantitative real-time polymerase chain reaction or Western blot. TYMS was high-expressed yet miR-140-3p was low-expressed in LUAD cells. Upregulation of miR-140-3p inhibited TYMS expression, viability, colony formation, migration, invasion, and tube length within LUAD cells, while downregulation of miR-140-3p did oppositely. Silenced TYMS, the downstream target gene of miR-140-3p, reversed the effects of miR-140-3p downregulation on TYMS expression, cell viability, colony formation, migration, invasion, and tube length as well as the metastasis-, apoptosis- and angiogenesis-related proteins in LUAD cells. Upregulation of miR-140-3p inhibited the proliferation, migration, invasion and angiogenesis of LUAD cells via targeting TYMS.
研究领域:
肿瘤学
·
分子生物学
·
细胞生物学
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产品关联
DOI
PubMed
高影响因子
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