Direct microRNA detection with universal tagged probe and time-resolved fluorescence technology

microRNAs have emerged as the central player in gene expression regulation and have been considered as potent cancer biomarkers for early disease diagnosis. Direct microRNA detection without amplification and labeling is highly desired. Here we present a rapid, sensitive and selective microRNA detection method based on the base stacking hybridization coupling with time-resolved fluorescence technology. Other than planar microarrays, magnetic beads are used as reaction platforms. In this method, one universal tag is used to report all microRNA targets. Its specificity allows for discrimination between microRNAs differing by a single nucleotide, and between precursor and mature microRNAs. This method also provides a high sensitivity down to 20 fM. Moreover, the full protocol can be completed in about 3 h starting from total RNA.     

Label-free reflectometric interference microchip biosensor based on nanoporous alumina for detection of circulating tumour cells

In this report, a label-free reflectometric interference spectroscopy (RIfS) based microchip biosensor for the detection of circulating tumour cells (CTCs) is demonstrated. Highly ordered nanoporous anodic aluminium oxide (AAO) fabricated by electrochemical anodization of aluminium foil was used as the RIfS sensing platform. Biotinylated anti-EpCAM antibody that specifically binds to human cancer cells of epithelial origin such as pancreatic cancer cells (PANC-1) was covalently attached to the AAO surface through multiple surface functionalization steps. Whole blood or phosphate buffer saline spiked with low numbers of pancreatic cancer cells were successfully detected by specially designed microfluidic device incorporating an AAO RIfS sensor, without labour intensive fluorescence labelling and/or pre-enhancement process. Our results show that the developed device is capable of selectively detecting of cancer cells, within a concentrations range of 1000-100,000 cells/mL, with a detection limit of <1000 cells/mL, a response time of <5 min and sample volume of 50 μL of. The presented RIfS method shows considerable promise for translation to a rapid and cost-effective point-of-care diagnostic device for the detection of CTCs in patients with metastatic cancer.     

Fluorescence-assisted cytological testing (FACT): Ex Vivo viral method for enhancing detection of rare cancer cells in body fluids

Cytological analysis of body fluids is currently used for detecting cancer. The objective of this study was to determine if the herpes virus carrying an enhanced green fluorescent protein (EGFP) could detect rare cancer cells in body fluids against millions of normal cells. Human cancer cells suspended with normal murine cells were infected with NV1066 at a multiplicity of infection (MOI) of 0.5 and 1.0 for 18 h. Fluorescent microscopy and flow cytometry were used for EGFP detection of cancer cells. EGFP-expressing cells were confirmed as cancer cells with specific markers by immunohistochemistry staining. Limits of detection of cancer cells in body fluid were measured by serial dilutions. Applicability of technique was confirmed with samples from patients with malignant pleural effusions. NV1066 expressed EGFP in 111 human cancer cell lines detected by fluorescent microscopy at an MOI of 0.5. NV1066 selectively infected cancer cells and spared normal cells as confirmed by immunohistochemistry. Sensitivity of detecting fluorescent green cells was 92% (confidence interval [CI] 83% to 97%) at a ratio of 1 cancer cell to 1 million normal cells. EGFP-positive cells were detected by fluorescent microscopy in patients' malignant pleural effusion samples. Our data show proof of the concept that NV1066-induced EGFP expression allows detection of a single cancer cell against a background of 1 million normal cells. This method was demonstrated to be a reliable screening tool for human cancer cells in a suspension of normal murine cells as well as clinical specimens of malignant pleural effusions.     

Identifying Cancer Specific Functionally Relevant miRNAs from Gene Expression and miRNA-to-Gene Networks Using Regularized Regression

Identifying microRNA signatures for the different types and subtypes of cancer can result in improved detection, characterization and understanding of cancer and move us towards more personalized treatment strategies. However, using microRNA''s differential expression (tumour versus normal) to determine these signatures may lead to inaccurate predictions and low interpretability because of the noisy nature of miRNA expression data. We present a method for the selection of biologically active microRNAs using gene expression data and microRNA-to-gene interaction network. Our method is based on a linear regression with an elastic net regularization. Our simulations show that, with our method, the active miRNAs can be detected with high accuracy and our approach is robust to high levels of noise and missing information. Furthermore, our results on real datasets for glioblastoma and prostate cancer are confirmed by microRNA expression measurements. Our method leads to the selection of potentially functionally important microRNAs. The associations of some of our identified miRNAs with cancer mechanisms are already confirmed in other studies (hypoxia related hsa-mir-210 and apoptosis-related hsa-mir-296-5p). We have also identified additional miRNAs that were not previously studied in the context of cancer but are coherently predicted as active by our method and may warrant further investigation. The code is available in Matlab and R and can be downloaded on http://www.cs.toronto.edu/goldenberg/Anna_Goldenberg/Current_Research.html.     

Aberrant DNA methylation of microRNA genes in human breast cancer – a critical appraisal

Aberrant DNA methylation of regulatory sequences is a well-documented mechanism of functional deletion of genes with anti-tumourigenic properties including microRNAs. This review discusses the publications describing aberrant methylation of microRNA genes in human breast cancer cells. Among the anti-tumourigenic properties of epigenetically inactivated microRNA genes, the inhibition of proliferation and of epithelial-to-mesenchymal transition (EMT) are the best studied. Several studies are conceptually very interesting and present a comprehensive functional characterization of anti-tumorigenic microRNAs. The link between microRNA expression and gene methylation is not addressed directly by all studies and a number of studies are limited in their strength by not including primary breast cancer specimens or by analysing very small sets of primary human specimens. The publications cover a wide range of DNA methylation detection techniques, often making direct comparison of results challenging. Despite the identification and thorough characterization of many interesting candidates and functionally important microRNA genes affected by DNA methylation, the translation of microRNA gene methylation as a new biomarker into the daily routine practice has not yet worked out.     

MicroRNA在肿瘤发生发展和诊断中作用的研究进展

microRNA是一类由内源基因编码的长度约为18-25个核苷酸的非编码单链RNA分子,可以与靶基因mRNA的3'非编码区结合,通过降解靶m RNA或(和)抑制靶m RNA转录后翻译调节靶蛋白的生成,从而发挥其生物学作用。目前,在人体基因组内发现的microRNA已经超过2500多个,可能调节着人类1/3的基因,在维持正常干细胞功能、调控细胞增殖分化及恶性肿瘤发生过程中均起重要作用。既往的研究表明microRNA与基因之间相互调控的失衡导致肿瘤的发生。从分子水平上研究microRNA与肿瘤发生的关系,检测microRNA与肿瘤相关基因表达情况的改变,分析肿瘤组织和血清中microRNA表达量与肿瘤分型的关系,将有利于肿瘤的病因学研究,早期发现和肿瘤治疗及预后判断。本文主要就microRNA在肿瘤发生发展和诊断中作用的研究进展进行了综述。     

Salivary MicroRNA in Pancreatic Cancer Patients

Background

Pancreatic cancer is the fourth leading cause of cancer death in Western countries, with the lowest 1-year survival rate among commonly diagnosed cancers. Reliable biomarkers for pancreatic cancer diagnosis are lacking and are urgently needed to allow for curative surgery. As microRNA (miRNA) recently emerged as candidate biomarkers for this disease, we explored in the present pilot study the differences in salivary microRNA profiles between patients with pancreatic tumors that are not eligible for surgery, precancerous lesions, inflammatory disease or cancer-free patients as a potential early diagnostic tool.

Methods

Whole saliva samples from patients with pancreatic cancer (n = 7), pancreatitis (n = 4), IPMN (n = 2), or healthy controls (n = 4) were obtained during endoscopic examination. After total RNA isolation, expression of 94 candidate miRNAs was screened by q(RT)PCR using Biomark Fluidgm. Human-derived pancreatic cancer cells were xenografted in athymic mice as an experimental model of pancreatic cancer.

Results

We identified hsa-miR-21, hsa-miR-23a, hsa-miR-23b and miR-29c as being significantly upregulated in saliva of pancreatic cancer patients compared to control, showing sensitivities of 71.4%, 85.7%, 85,7% and 57%, respectively and excellent specificity (100%). Interestingly, hsa-miR-23a and hsa-miR23b are overexpressed in the saliva of patients with pancreatic cancer precursor lesions. We found that hsa-miR-210 and let-7c are overexpressed in the saliva of patients with pancreatitis as compared to the control group, with sensitivity of 100% and 75%, and specificity of 100% and 80%, respectively. Last hsa-miR-216 was upregulated in cancer patients as compared to patients diagnosed with pancreatitis, with sensitivity of 50% and specificity of 100%. In experimental models of PDAC, salivary microRNA detection precedes systemic detection of cancer cells markers.

Conclusions

Our novel findings indicate that salivary miRNA are discriminatory in pancreatic cancer patients that are not eligible for surgery. In addition, we demonstrate in experimental models that salivary miRNA detection precedes systemic detection of cancer cells markers. This study stems for the use of salivary miRNA as biomarker for the early diagnosis of patients with unresectable pancreatic cancer.     

SC-BPSO:肝癌分类中一种融合过滤器的二进制粒子群算法特征的选择方法

癌症的早期诊断能够显著提高癌症患者的存活率,在肝细胞癌患者中这种情况更加明显。机器学习是癌症分类中的有效工具。如何在复杂和高维的癌症数据集中,选择出低维度、高分类精度的特征子集是癌症分类的难题。本文提出了一种二阶段的特征选择方法SC-BPSO:通过组合Spearman相关系数和卡方独立检验作为过滤器的评价函数,设计了一种新型的过滤器方法——SC过滤器,再组合SC过滤器方法和基于二进制粒子群算法(BPSO)的包裹器方法,从而实现两阶段的特征选择。并应用在高维数据的癌症分类问题中,区分正常样本和肝细胞癌样本。首先,对来自美国国家生物信息中心(NCBI)和欧洲生物信息研究所(EBI)的130个肝组织microRNA序列数据(64肝细胞癌,66正常肝组织)进行预处理,使用MiRME算法从原始序列文件中提取microRNA的表达量、编辑水平和编辑后表达量3类特征。然后,调整SC-BPSO算法在肝细胞癌分类场景中的参数,选择出关键特征子集。最后,建立分类模型,预测结果,并与信息增益过滤器、信息增益率过滤器、BPSO包裹器特征选择算法选出的特征子集,使用相同参数的随机森林、支持向量机、决策树、KNN四种分类器分类,对比分类结果。使用SC-BPSO算法选择出的特征子集,分类准确率高达98.4%。研究结果表明,与另外3个特征选择算法相比,SC-BPSO算法能有效地找到尺寸较小和精度更高的特征子集。这对于少量样本高维数据的癌症分类问题可能具有重要意义。     

Selection of microRNA for providing tumor specificity of transgene expression in cancer gene therapy

The use of tumor-specific microRNA loss to inhibit transgene expression in normal cells is considered as a way to increase the specificity of gene-therapeutic antitumor drugs. This method assumes the introduction of recognition sites of suppressed in tumor cells microRNAs into transgene transcipt. In the presented work, the efficiency of the strategy for providing the tumor specificity of transgene expression depending on parameters of microRNA expression in normal and tumor cells was studied. It was established that microRNA suppression in tumor cells and the determination of absolute microRNA levels in tumor and normal cells are not sufficient for the adequate estimation of the possibility of specific microRNA usage in the scheme of cancer gene therapy, and particularly do not allow to exclude a significant decrease in the efficiency of the gene-therapeutic drug upon the introduction of microRNA recognition sites. These parameters are only suitable for the preliminary selection of microRNA. The effect of introduction of microRNA recognition sites on transgene expression level in target tumor cells should be validated experimentally. It is suggested that this should be done directly in the cancer gene therapy scheme with monitoring of the therapeutic transgene activity.     

Amplified microRNA detection by templated chemistry

MicroRNAs (miRNAs) are a class of RNAs that play important regulatory roles in the cell. The detection of microRNA has attracted significant interest recently, as abnormal miRNA expression has been linked to cancer and other diseases. Here, we present a straightforward method for isothermal amplified detection of miRNA that involves two separate nucleic acid-templated chemistry steps. The miRNA first templates the cyclization of an oligodeoxynucleotide from a linear precursor containing a 5'-iodide and a 3'-phosphorothioate. The sequence is amplified through rolling circle amplification with 29 DNA polymerase and then detected via a second amplification using fluorogenic templated probes. Tests showed that the cyclization proceeds in ～50% yield over 24 h and is compatible with the conditions required for rolling circle polymerization, unlike enzymatic ligations which required non-compatible buffer conditions. The polymerization yielded 188-fold amplification, and separate experiments showed ～15-fold signal amplification from the templated fluorogenic probes. When all components are combined, results show miRNA detection down to 200 pM in solution, and correlation of the detected signal with the initial concentration of miRNA. The doubly templated double-amplification method demonstrates a new approach to detection of rolling circle products and significant advantages in ease of operation for miRNA detection.     

MicroRNA Expression Profiles of Whole Blood in Lung Adenocarcinoma

The association of lung cancer with changes in microRNAs in plasma shown in multiple studies suggests a utility for circulating microRNA biomarkers in non-invasive detection of the disease. We examined if presence of lung cancer is reflected in whole blood microRNA expression as well, possibly because of a systemic response. Locked nucleic acid microarrays were used to quantify the global expression of microRNAs in whole blood of 22 patients with lung adenocarcinoma and 23 controls, ten of whom had a radiographically detected non-cancerous lung nodule and the other 13 were at high risk for developing lung cancer because of a smoking history of >20 pack-years. Cases and controls differed significantly for age with a mean difference of 10.7 years, but not for gender, race, smoking history, blood hemoglobin, platelet count, or white blood cell count. Of 1282 quantified human microRNAs, 395 (31%) were identified as expressed in the study’s subjects, with 96 (24%) differentially expressed between cases and controls. Classification analyses of microRNA expression data were performed using linear kernel support vector machines (SVM) and top-scoring pairs (TSP) methods, and classifiers to identify presence of lung adenocarcinoma were internally cross-validated. In leave-one-out cross-validation, the TSP classifiers had sensitivity and specificity of 91% and 100%, respectively. The values with SVM were both 91%. In a Monte Carlo cross-validation, average sensitivity and specificity values were 86% and 97%, respectively, with TSP, and 88% and 89%, respectively, with SVM. MicroRNAs miR-190b, miR-630, miR-942, and miR-1284 were the most frequent constituents of the classifiers generated during the analyses. These results suggest that whole blood microRNA expression profiles can be used to distinguish lung cancer cases from clinically relevant controls. Further studies are needed to validate this observation, including in non-adenocarcinomatous lung cancers, and to clarify upon the confounding effect of age.     

Epigenetic regulation of prostate cancer

Prostate cancer is a commonly diagnosed cancer in men and a leading cause of cancer deaths. Whilst the underlying mechanisms leading to prostate cancer are still to be determined, it is evident that both genetic and epigenetic changes contribute to the development and progression of this disease. Epigenetic changes involving DNA hypo- and hypermethylation, altered histone modifications and more recently changes in microRNA expression have been detected at a range of genes associated with prostate cancer. Furthermore, there is evidence that particular epigenetic changes are associated with different stages of the disease. Whilst early detection can lead to effective treatment, and androgen deprivation therapy has a high response rate, many tumours develop towards hormone-refractory prostate cancer, for which there is no successful treatment. Reliable markers for early detection and more effective treatment strategies are, therefore, needed. Consequently, there is a considerable interest in the potential of epigenetic changes as markers or targets for therapy in prostate cancer. Epigenetic modifiers that demethylate DNA and inhibit histone deacetylases have recently been explored to reactivate silenced gene expression in cancer. However, further understanding of the mechanisms and the effects of chromatin modulation in prostate cancer are required. In this review, we examine the current literature on epigenetic changes associated with prostate cancer and discuss the potential use of epigenetic modifiers for treatment of this disease.     

Differential MicroRNA Regulation Correlates with Alternative Polyadenylation Pattern between Breast Cancer and Normal Cells

Alternative polyadenylation (APA) could result in mRNA isoforms with variable lengths of 3′ UTRs. Gain of microRNA target sites in the 3′ UTR of a long mRNA isoform may cause different regulation from the corresponding short isoform. It has been known that cancer cells globally exhibit a lower ratio of long and short isoforms (LSR); that is, they tend to express larger amounts of short isoforms. The objective of this study is to illustrate the relationship between microRNA differential regulation and LSR. We retrieved public APA annotations and isoform expression profiles of breast cancer and normal cells from a high-throughput sequencing method study specific for the mRNA 3′ end. Combining microRNA expression profiles, we performed statistical analysis to reveal and estimate microRNA regulation on APA patterns in a global scale. First, we found that the amount of microRNA target sites in the alternative UTR (aUTR), the region only present in long isoforms, could affect the LSR of the target genes. Second, we observed that the genes whose aUTRs were targeted by up-regulated microRNAs in cancer cells had an overall lower LSR. Furthermore, the target sites of up-regulated microRNAs tended to appear in aUTRs. Finally, we demonstrated that the amount of target sites for up-regulated microRNAs in aUTRs correlated with the LSR change between cancer and normal cells. The results indicate that up-regulation of microRNAs might cause lower LSRs of target genes in cancer cells through degradation of their long isoforms. Our findings provide evidence of how microRNAs might play a crucial role in APA pattern shifts from normal to cancerous or proliferative states.     

Genome-wide prioritization of disease genes and identification of disease-disease associations from an integrated human functional linkage network

Background

Recent studies have shown that the regulatory effect of microRNAs can be investigated by examining expression changes of their target genes. Given this, it is useful to define an overall metric of regulatory effect for a specific microRNA and see how this changes across different conditions.

Results

Here, we define a regulatory effect score (RE-score) to measure the inhibitory effect of a microRNA in a sample, essentially the average difference in expression of its targets versus non-targets. Then we compare the RE-scores of various microRNAs between two breast cancer subtypes: estrogen receptor positive (ER⁺) and negative (ER^-). We applied this approach to five microarray breast cancer datasets and found that the expression of target genes of most microRNAs was more repressed in ER^- than ER⁺; that is, microRNAs appear to have higher RE-scores in ER^- breast cancer. These results are robust to the microRNA target prediction method. To interpret these findings, we analyzed the level of microRNA expression in previous studies and found that higher microRNA expression was not always accompanied by higher inhibitory effects. However, several key microRNA processing genes, especially Ago2 and Dicer, were differentially expressed between ER^- and ER⁺ breast cancer, which may explain the different regulatory effects of microRNAs in these two breast cancer subtypes.

Conclusions

The RE-score is a promising indicator to measure microRNAs' inhibitory effects. Most microRNAs exhibit higher RE-scores in ER^- than in ER⁺ samples, suggesting that they have stronger inhibitory effects in ER^- breast cancers.     

Improved northern blot method for enhanced detection of small RNA

This protocol describes an improved northern blot method that enhances detection of small RNA molecules (<40 nt) including regulatory species such as microRNA (miRNA), short-interfering RNA (siRNA) and Piwi-interacting RNA. Northern blot analysis involves the separation of RNA molecules by denaturing gel electrophoresis followed by transfer and cross-linking of the separated molecules to nylon membrane. RNA of interest is then detected by hybridization with labeled complementary nucleic acid probes. We have replaced conventional UV-cross-linking of RNA to nylon membranes with a novel, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)-mediated, chemical cross-linking step that enhances detection of small RNA by up to 50-fold. This requires no specialized equipment, is relatively inexpensive and is technically straightforward. Northern blotting can be done in 2 d, but detection of a specific RNA can vary from minutes to days. Although chemical cross-linking takes longer (15 min to 2 h) than UV cross-linking, improved sensitivity means shorter periods of exposure are required to detect signal after hybridization.