Primary microcephaly gene MCPH1 shows a novel molecular biomarker of human renal carcinoma and is regulated by miR-27a

Microcephalin 1 (MCPH1) gene, initially identified as an hTERT repressor, result in two autosomal recessive disorders: primary microcephaly and premature chromosome condensation syndrome. Recently, several studies have found that MCPH1 has also been shown to be downregulated in several different types of human cancers, suggesting that it could also function as a tumor suppressor gene and a novel molecular biomarker of human cancers. To investigate its potential role in the human renal carcinoma progression, we evaluated the expression of protein MCPH1 in 188 renal cancer and 20 normal renal tissues from 188 patients with renal cancer and 20 healthy persons by immunohistochemistry. Positive MCPH1 staining was found in all normal renal samples and partly in cancerous tissues. But MCPH1-positive cells resulted significantly lower in renal carcinoma tissues compared with normal tissues. We further observed that overexpression of MCPH1 decreased cellular proliferation, cell migration and invasion and induced cell apoptosis, indicating it is tumor suppressor. Using bioinformatics approaches and luciferase reporter assay, we showed that the 3’-UTR of MCPH1 harbors two non-overlapping functional seed regions for miR-27 which negatively regulated its level. The expression level of miR-27a negatively correlated with the MCPH1 protein level in renal cancer. Our study indicates for the first time that, in addition to its role in brain development, MCPH1 also functions as a tumor suppressor gene and is directly regulated by miR-27a.     

Increased expression of miR-24 is associated with acute myeloid leukemia with t(8;21)

This study was designed to learn the expression status of miR-24 and its clinical relevance in patients with acute myeloid leukemia (AML). We detected the miR-24 expression levels using real-time quantitative PCR in 84 AML patients and investigated the clinical significance of miR-24 expression in AML. There was no difference in clinical parameters between cases with miR-24 high expression and with miR-24 low expression. The frequency of miR-24 high expression was higher in patients with t(8;21) than in others (82% (9/11) versus 44% (32/72), P=0.026). The levels of miR-24 expression had no correlation with the mutations of nine genes (FLT3-ITD, NPM1, C-KIT, IDH1/IDH2, DNMT3A, N/K-RAS and C/EBPA). Meanwhile, among the group who obtained CR, the cases with miR-24 high expression had no difference in overall survival (OS) and relapse-free survival (RFS) than those with miR-24 low expression (P=0.612 and 0.665, respectively). These findings implicated that miR-24 high regulation is a common event in AML with t(8;21), and it might serve as a novel and selective therapeutic target for the treatment of AML with t(8;21).     

Exploring the role of the complement system,endothelial injury,and microRNAs in thrombotic microangiopathy after kidney transplantation

ObjectiveWe investigated whether the recipient’s complement system function, kidney graft endothelial ultrastructural injury, and microRNA (miRNA) expression before transplantation may be associated with the risk of posttransplant de novo thrombotic microangiopathy (TMA).MethodsComplement system function assessment, histological and ultrastructural examination of preimplantation and kidney graft biopsies, and microRNA assessment were performed on kidney transplant recipients (KTRs) with de novo TMA.ResultsOn the basis of the clinical course, histological findings, and miRNA patterns, the following two de novo TMA phenotypes were observed: a self-limiting disease that was localized to the kidney graft and a systemic disease that progressed to graft failure without timely treatment. Decreased alternative complement pathway activity and ultrastructural endothelial injury before transplantation were confirmed in all five KTRs and four of five KTRs, respectively, but they did not correlate with de novo TMA severity.ConclusionsAlternative complement pathway abnormalities in KTRs and endothelial ultrastructural injury on preimplantation biopsy might be associated with de novo posttransplant TMA, although they did not predict posttransplant TMA severity (localized vs. systemic). The specific miRNA expression patterns in preimplantation kidney graft biopsies demonstrated a borderline statistically significant difference and might provide more accurate information on posttransplant TMA severity.     

血清微小RNA Let-7a表达水平对儿童哮喘诊断及严重程度评估的价值

目的研究哮喘儿童循环血清微小RNA Let-7a的表达水平,探讨其在哮喘诊断及疾病严重程度评估中的价值。方法本研究纳入90例哮喘儿童(哮喘组)和90例健康儿童(健康对照组)作为研究对象,采用酶联免疫吸附试验法检测研究对象血清中白细胞介素(IL)-13水平,所有研究对象均接受肺功能检测,通过实时荧光定量PCR测量血浆Let-7a表达水平。采用受试者工作特征曲线(ROC曲线)分析血清Let-7a对儿童哮喘的诊断价值及其区分重度和中轻度哮喘儿童的能力,采用Pearson线性相关分析血清Let-7a与哮喘儿童肺功能参数和血清IL-13的相关性。结果哮喘组儿童血清Let-7a表达水平显著低于健康对照组儿童(P=0.000),哮喘组儿童血清IL-13水平显著高于健康对照组儿童(P=0.000);哮喘组儿童血清Let-7a表达水平随着哮喘严重程度显著下降(P=0.000)。当最佳临界值为2.09,血清Let-7a诊断哮喘儿童的ROC曲线下面积为0.92(95%CI:0.87~0.95,P=0.001),灵敏度和特异度分别为85.0%和90.0%。当最佳临界值为1.60,Let-7a区分重度和中轻度哮喘的ROC曲线下面积为0.80(95%CI:0.68~0.92,P=0.001),灵敏度和特异度分别为75.0%和93.5%。Let-7a表达水平与IL-13水平呈负相关(r=-0.31,P=0.001),Let-7a表达水平与第1秒用力呼气容积和用力肺活量均呈正相关(r=0.270,P=0.004;r=0.412,P=0.002)。结论Let-7a可用作血清非侵入性标志物,用于诊断哮喘及其严重程度。     

基于甲状腺髓样癌分子病理机制的基因诊断研究进展

甲状腺髓样癌(medullary thyroid carcinoma,MTC)是一种罕见的恶性神经内分泌肿瘤,具有强侵袭性和较差的预后性。由于传统的影像学和病理学检查的局限性,MTC患者的确诊通常已处于晚期,因此亟待开发准确灵敏的MTC早期诊断技术来协助临床治疗。近年来,基于MTC分子病理机制的基因诊断技术已经引起了人们广泛的关注,尤其是多种微小RNA(microRNA,miRNA)在MTC中的差异性表达和基因调节作用,使得其具有潜在的早期诊断、精准预后和基因治疗等重大意义。     

Dynamic single-molecule sensing by actively tuning binding kinetics for ultrasensitive biomarker detection

The ability to measure many single molecules simultaneously in large and complex samples is critical to the translation of single-molecule sensors for practical applications in biomarker detection. The challenges lie in the limits imposed by mass transportation and thermodynamics, resulting in long assay time and/or insufficient sensitivity. Here, we report an approach called Sensing Single Molecule under MicroManipulation (SSM³) to circumvent the above limits. In SSM³, single-molecule binding processes were dynamically recorded by surface plasmon resonance microscopy in a nanoparticle-mediated sandwich scheme. The binding kinetics between analyte and probes are fine-tuned by nanoparticle micromanipulations to promote the repetitive binding and dissociation. Quantifying the heterogeneous lifetime of each molecular complex allows the discrimination of specific binding from nonspecific background noise. By digitally counting the number of repetitive specific binding events, we demonstrate the direct detection of microRNAs and amyloid-β proteins with the limit of detection at the subfemtomolar level in buffer and spiked human serum. Together with the nanoparticle micromanipulation to promote the transportation rate of analyte molecules, the assay could be performed within as short as 15 min without the need for preincubation. The advantages over other single-molecule sensors include short assay time, compatible with common probes and ultrasensitive detection. With further improvement on the throughput and automation, we anticipate the proposed approach could find wide applications in fundamental biological research and clinical testing of disease-related biomarkers.

The analytical methods have converged from ensemble measurements of numerous entities to quantized measurements at the single-molecule level. Single-molecule measurements could reveal heterogeneities and stochastic processes within biological systems (1, 2) and set the ultimate detection limit of chemical and biological sensors. By reducing the measurement volume to a few femtoliters, the detection of a single molecule has been realized in various forms [i.e., single-molecule fluorescence (3, 4), nanopores (5, 6), localized surface plasmon resonance (7, 8), and surface-enhanced Raman scattering (9, 10)]. These measurements typically require quantifying many single-molecule events to gain new molecular and mechanistic insights or to achieve better analytical performance. However, it has been difficult to perform quantitative analysis with sufficient efficiency and statistical accuracy because of the concentration limit from mass transportation (11, 12) and the thermodynamic limit from probe affinity (13). For quantification of biomarkers in biological media, in which the required concentrations are usually at the femtomolar level or even lower (14), the single-molecule measurements could take inordinately long, and the nonspecific binding of unwanted species degrades the accuracy.In the past two decades, several single-molecule approaches for biomarker detection have been developed to surpass the above limits by biasing the equilibrium and driving binding reactions (15, 16). A typical scheme involves the usage of nanoparticles to collect the analyte followed by a digital measurement of single molecules at a confined space (17), such as the commercialized, single-molecule enzyme-linked immunosorbent analysis (digital ELISA) (18). The digital ELISA uses the antibody-modified magnetic beads to capture the analyte in solution and loads them into femtoliter-sized reaction chambers termed single-molecule arrays. It effectively improves the sensitivity of conventional ELISA by three orders with a limit of detection (LoD) at the subfemtomolar level but requires sophisticated devices and excessive operation to remove free analyte molecules. Besides, the performance is still limited by the probe affinity and false positive arising from detection antibodies that bind nonspecifically to assay surface.A distinct yet effective strategy is to explore the in-depth heterogeneous information of single-molecule interaction (19). Walter et al. first demonstrated a kinetic fingerprinting approach to perform highly specific and sensitive detection of biomarkers via single-molecule fluorescence microscopy (20–22). This single-molecule recognition through equilibrium Poisson sampling technique surpasses the thermodynamic limit by exploiting the repetitive binding of fluorescently labeled, low-affinity probes to the analyte (23) and discriminating specific binding from background noise by a kinetic signature. The detection limits of microRNAs (miRNAs) and proteins also reach the subfemtomolar level, but screening probes with unique kinetic property is not compatible with current pipelines, and the concentration limit implies long incubation time before detection.Herein, we present the integration of single-molecule manipulation and dynamic sensing to allow rapid and ultrasensitive detection of biomarkers beyond the concentration and thermodynamic limits. In this Sensing Single Molecule under MicroManipulation (SSM³) approach, an external force is applied on the molecular bound between analyte and probes through tethered nanoparticles to actively tune the binding kinetics. This strategy, together with a dynamic sensing approach to exploit the heterogeneity at the single-molecule level, is able to beat the limits in both assay time and sensitivity. We show the principle and realization of the SSM³ technique and demonstrate 15-min assays to directly measure miRNAs and proteins at the subfemtomolar concentration.     

微小RNA甲基化在肝细胞癌诊疗中的研究进展

微小RNA(microRNA,miRNA)是一类在转录后水平调节基因表达的非编码短链RNA,在细胞的生命活动中具有多种调节作用。甲基化是从基因组层面研究RNA转录调控的重要领域,通过调节基因的表达和沉默,与肿瘤、衰老等许多疾病密切相关。研究表明,miRNA甲基化异常可以调控miRNA的表达,并影响其靶基因的表达和功能,是肝细胞癌发生发展的关键信号,这为进一步研究肝细胞癌发病的分子机制和探索有价值的治疗靶点提供新的思路。