An integrated nano-scale approach to profile miRNAs in limited clinical samples

Profiling miRNA expression in cells that directly contribute to human disease pathogenesis is likely to aid the discovery of novel drug targets and biomarkers. However, tissue heterogeneity and the limited amount of human diseased tissue available for research purposes present fundamental difficulties that often constrain the scope and potential of such studies. We established a flow cytometry-based method for isolating pure populations of pathogenic T cells from bronchial biopsy samples of asthma patients, and optimized a high-throughput nano-scale qRT-PCR method capable of accurately measuring 96 miRNAs in as little as 100 cells. Comparison of circulating and airway T cells from healthy and asthmatic subjects revealed asthma-associated and tissue-specific miRNA expression patterns. These results establish the feasibility and utility of investigating miRNA expression in small populations of cells involved in asthma pathogenesis, and set a precedent for application of our nano-scale approach in other human diseases. The microarray data from this study (Figure 7) has been submitted to the NCBI Gene Expression Omnibus (GEO; http://ncbi.nlm.nih.gov/geo) under accession no. GSE31030.

miR-15a and miR-16 are implicated in cell cycle regulation in a Rb-dependent manner and are frequently deleted or down-regulated in non-small cell lung cancer

MicroRNAs (miRNA) are negative regulators of gene expression at the posttranscriptional level, which are involved in tumorigenesis. Two miRNAs, miR-15a and miR-16, which are located at chromosome 13q14, have been implicated in cell cycle control and apoptosis, but little information is available about their role in solid tumors. To address this question, we established a protocol to quantify miRNAs from laser capture microdissected tissues. Here, we show that miR-15a/miR-16 are frequently deleted or down-regulated in squamous cell carcinomas and adenocarcinomas of the lung. In these tumors, expression of miR-15a/miR-16 inversely correlates with the expression of cyclin D1. In non-small cell lung cancer (NSCLC) cell lines, cyclins D1, D2, and E1 are directly regulated by physiologic concentrations of miR-15a/miR-16. Consistent with these results, overexpression of these miRNAs induces cell cycle arrest in G(1)-G(0). Interestingly, H2009 cells lacking Rb are resistant to miR-15a/miR-16-induced cell cycle arrest, whereas reintroduction of functional Rb resensitizes these cells to miRNA activity. In contrast, down-regulation of Rb in A549 cells by RNA interference confers resistance to these miRNAs. Thus, cell cycle arrest induced by these miRNAs depends on the expression of Rb, confirming that G(1) cyclins are major targets of miR-15a/miR-16 in NSCLC. Our results indicate that miR-15a/miR-16 are implicated in cell cycle control and likely contribute to the tumorigenesis of NSCLC.

Distinct microRNA Expression Profile in Prostate Cancer Patients with Early Clinical Failure and the Impact of let-7 as Prognostic Marker in High-Risk Prostate Cancer

Background The identification of additional prognostic markers to improve risk stratification and to avoid overtreatment is one of the most urgent clinical needs in prostate cancer (PCa). MicroRNAs, being important regulators of gene expression, are promising biomarkers in various cancer entities, though the impact as prognostic predictors in PCa is poorly understood. The aim of this study was to identify specific miRNAs as potential prognostic markers in high-risk PCa and to validate their clinical impact. Methodology and Principal Findings We performed miRNA-microarray analysis in a high-risk PCa study group selected by their clinical outcome (clinical progression free survival (CPFS) vs. clinical failure (CF)). We identified seven candidate miRNAs (let-7a/b/c, miR-515-3p/5p, -181b, -146b, and -361) that showed differential expression between both groups. Further qRT-PCR analysis revealed down-regulation of members of the let-7 family in the majority of a large, well-characterized high-risk PCa cohort (n = 98). Expression of let-7a/b/and -c was correlated to clinical outcome parameters of this group. While let-7a showed no association or correlation with clinical relevant data, let-7b and let-7c were associated with CF in PCa patients and functioned partially as independent prognostic marker. Validation of the data using an independent high-risk study cohort revealed that let-7b, but not let-7c, has impact as an independent prognostic marker for BCR and CF. Furthermore, we identified HMGA1, a non-histone protein, as a new target of let-7b and found correlation of let-7b down-regulation with HMGA1 over-expression in primary PCa samples. Conclusion Our findings define a distinct miRNA expression profile in PCa cases with early CF and identified let-7b as prognostic biomarker in high-risk PCa. This study highlights the importance of let-7b as tumor suppressor miRNA in high-risk PCa and presents a basis to improve individual therapy for high-risk PCa patients.

E2F1 AND TUMOR SUPPRESSION: THE ROLE OF p21, miRNAS, AND THE DNA DAMAGE RESPONSE

E2F1 is a multi-faceted protein that has roles in a number of important cellular processes including cell cycle regulation, apoptosis, proliferation, and the DNA damage response (DDR). Moreover, E2F1 has opposing roles in tumor development, acting as either a tumor suppressor or an oncogene depending on the context. In human cancer, E2F1 is often deregulated through aberrations in the Rb-p16INK4a-cyclin D1 pathway. In these studies we examined three mechanisms by which E2F1 might mediate its tumor suppressive properties: p21-induced senescence, miRNAs, and the DNA damage response. We found that E2F1 acts as a tumor suppressor in response to ras activation through a non-apoptotic mechanism requiring ARF and p53, but not p21. However, p21-loss inhibited two-stage chemical carcinogenesis in FVB mice. In response to E2F1 overexpression, we found that 22 miRNAs are differentially regulated in mouse epidermis, including let-7a, let-7c, and miR-301. Additionally, regulation of miR-301 involves binding of E2F1 to its promoter. Finally, our data indicate a role for E2F1 at sites of DNA damage requiring E2F1’s phosphorylation at serine 31 which may involve DNA repair. Further, this role in the DDR may affect tumor aggressiveness and multiplicity. In all, we have explored three mechanisms for E2F1-induced tumor suppression and identified E2F1’s role in the DNA damage response as a likely contributor to this phenomenon.

Novel expression of microRNAs in serum samples of Iraqi breast cancer women

Although a lot of hard work against cancer to reduces its spread but it still continues to kill with abandon. The need for a biomarker for cancer early detection becomes the most mind concentrated scientists. MicroRNAs the tiny non coding RNA molecules opened new path for the scientists to determine the cancer in its early stages. Expression of microRNAs profiles has been investigated to be involved in cancer development. Here we determined the expression of microRNAs in serum of Iraqi healthy volunteers and other women diagnosed with breast cancer. MicroRNAs expression has been determined by using real time qPCR and delta method has been used. Four of thirteen microRNAs were shown to be expressed in serum of Iraqi breast cancer women. Let-7a and miR-21 were shown to be significantly over expressed in serum of breast cancer compared with healthy serum volunteers (P= 0.022 and 0.026) respectively. While miR-26b and miR-429 found to be significantly down expressed in serum of breast cancer women (P= 0.0034 and 0.031) respectively. The result concluded that these expressed microRNAs in serum of breast cancer women could be used as a first indicator of breast cancer occurrence.

miRNAS circulantes como biomarcadores para fibrilação atrial aguda

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Some non-coding RNAs (miRNAs) have been involved in regulatory activity in arrhythmogenesis, targeting genes that contribute to the development of AF. The present study aimed to evaluate the expression of candidate miRNAs in plasma from patients with AF and new-onset AF and its application as future markers for diagnosis and monitoring of disease. miR-21, miR-133a, miR-133b, miR-150, miR-328 and miR-499 were selected as targets in this study through a prior literature review. They were isolated from plas-ma of individuals aged from 20 to 85 years old with AF (n = 17), new-onset AF (n = 5) and without AF (n = 15), where the latter was the control group. The results were ana-lyzed by Real-Time PCR (RT-PCR) with miScript SYBR Green PCR. We observed that miR-21, miR-133b, miR-328 and miR-499 had different levels of expression be-tween the three groups (p <0.05). Increased expression of miR-21 (0.6-fold), miR-133b (1.4-fold), miR-328 (2.0-fold) and miR-499 (2.3-fold) in patients with new-onset AF when compared to AF and control subjects. The miR-133a and miR-150 expression did not differ among the groups. miR-21, miR-133b, miR-328 and miR-499 may be potential biomarkers for AF as well as for new-onset AF, for monitoring and for the di-agnosis. These findings may contribute to the understanding of the process that trig-gers AF and suggest application these molecules as future biomarkers for AF.

miRNAS circulantes como biomarcadores para fibrilação atrial aguda

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Some non-coding RNAs (miRNAs) have been involved in regulatory activity in arrhythmogenesis, targeting genes that contribute to the development of AF. The present study aimed to evaluate the expression of candidate miRNAs in plasma from patients with AF and new-onset AF and its application as future markers for diagnosis and monitoring of disease. miR-21, miR-133a, miR-133b, miR-150, miR-328 and miR-499 were selected as targets in this study through a prior literature review. They were isolated from plas-ma of individuals aged from 20 to 85 years old with AF (n = 17), new-onset AF (n = 5) and without AF (n = 15), where the latter was the control group. The results were ana-lyzed by Real-Time PCR (RT-PCR) with miScript SYBR Green PCR. We observed that miR-21, miR-133b, miR-328 and miR-499 had different levels of expression be-tween the three groups (p <0.05). Increased expression of miR-21 (0.6-fold), miR-133b (1.4-fold), miR-328 (2.0-fold) and miR-499 (2.3-fold) in patients with new-onset AF when compared to AF and control subjects. The miR-133a and miR-150 expression did not differ among the groups. miR-21, miR-133b, miR-328 and miR-499 may be potential biomarkers for AF as well as for new-onset AF, for monitoring and for the di-agnosis. These findings may contribute to the understanding of the process that trig-gers AF and suggest application these molecules as future biomarkers for AF.

MicroRNA signatures differentiate preserved from reduced ejection fraction heart failure

AIMS: Differentiation of heart failure with reduced (HFrEF) or preserved (HFpEF) ejection fraction independent of echocardiography is challenging in the community. Diagnostic strategies based on monitoring circulating microRNA (miRNA) levels may prove to be of clinical value in the near future. The aim of this study was to identify a novel miRNA signature that could be a useful HF diagnostic tool and provide valuable clinical information on whether a patient has HFrEF or HFpEF.

METHODS AND RESULTS: MiRNA biomarker discovery was carried out on three patient cohorts, no heart failure (no-HF), HFrEF, and HFpEF, using Taqman miRNA arrays. The top five miRNA candidates were selected based on differential expression in HFpEF and HFrEF (miR-30c, -146a, -221, -328, and -375), and their expression levels were also different between HF and no-HF. These selected miRNAs were further verified and validated in an independent cohort consisting of 225 patients. The discriminative value of BNP as a HF diagnostic could be improved by use in combination with any of the miRNA candidates alone or in a panel. Combinations of two or more miRNA candidates with BNP had the ability to improve significantly predictive models to distinguish HFpEF from HFrEF compared with using BNP alone (area under the receiver operating characteristic curve >0.82).

CONCLUSION: This study has shown for the first time that various miRNA combinations are useful biomarkers for HF, and also in the differentiation of HFpEF from HFrEF. The utility of these biomarker combinations can be altered by inclusion of natriuretic peptide. MiRNA biomarkers may support diagnostic strategies in subpopulations of patients with HF.

Cross-regulation between cytokine and microRNA pathways in T cells

microRNA (miRNA) mediated regulation of protein expression has emerged as an important mechanism in T-cell physiology, from development and survival to activation, proliferation, and differentiation. One of the major classes of proteins involved in these processes are cytokines, which are both key input signals and major products of T-cell function. Here, we summarize the current data on the molecular cross-talk between cytokines and miRNAs: how cytokines regulate miRNA expression, and how specific miRNAs control cytokine production in T cells. We also describe the inflammatory consequences of deregulating the miRNA/cytokine axis in mice and humans. We believe this topical area will have key implications for immune modulation and treatment of autoimmune pathology.

The role of miRNAs in stroke

Stroke is currently one of the leading causes of death and disability worldwide. Despite recent advances in the treatment of stroke there is a major unmet clinical need for novel therapeutics for intervention. miRNAs are small coding RNAs which act to post-transcriptionally inhibit expression of genes. Emerging evidence has supported the view that miRNAs play an important role in the development and progression of ischaemic stroke, although understanding remains relatively poor. This research uses several models to investigate the effects of miRNAs in the context of stroke in vivo and in vitro, as well as assessment of patient serum samples in order to identify biomarkers for stroke. miR-29b was found to be significantly upregulated in SHRSP rat brain peri-infarct at 72h following stroke, and downregulated in ischaemic core at 24h and 72h following stroke, whilst miR-29c was significantly downregulated in remainder tissue at 24h following stroke and in infarct at 72h following stroke. The upreglation of miR-29b at 72h corresponded to a significant downregulation of miR-29 target genes MMP2, MMP9 and TGF-β1 in peri-infarct tissue at 72h following stroke. Modulation of miR-29b and miR-29c was achieved in a rat neuronal cell line but suppression of genes of interest was not observed following oxygen glucose deprivation. Several candidate miRNAs were then identified by microRNA Openarray analysis in stroke patient serum samples. Validation of these miRNAs was not demonstrated in the population studied, but assessment of these miRNAs in rat serum and isolated exosomes demonstrated that several of these miRNAs were significantly altered in SHRSP rats following stroke. Finally miR-21 was demonstrated to be significantly upregulated in SHRSP rat peri-infarct following stroke. This was associated with a change in miR-21 localization as determined by in situ hybridization. Modulation of miR-21 via the use of CAG-miR-21 mice demonstrated no difference in infarct size as measured by T2 -weighted MRI scan nor was any difference present in behavioural tests versus wild type. KO of miR-21 resulted in a reduction of survival rate compared with wild type. This thesis demonstrates that miR-29 and miR-21 are modulated following stroke in animal models, and these are potential candidates for therapeutic intervention in the future. Analysis of clinical samples has illustrated difficulties in the identification of serum miRNA profiles and suggests that looking at the exosomal component of serum may provide better information regarding miRNA profiles after stroke.