Platelets in patients with premature coronary artery disease exhibit upregulation of miRNA340* and miRNA624*

Background

Coronary artery disease (CAD) is the leading cause of human morbidity and mortality worldwide, underscoring the need to improve diagnostic strategies. Platelets play a major role, not only in the process of acute thrombosis during plaque rupture, but also in the formation of atherosclerosis itself. MicroRNAs are endogenous small non-coding RNAs that control gene expression and are expressed in a tissue and disease-specific manner. Therefore they have been proposed to be useful biomarkers. It remains unknown whether differences in miRNA expression levels in platelets can be found between patients with premature CAD and healthy controls.

Methodology/Principal Findings

In this case-control study we measured relative expression levels of platelet miRNAs using microarrays from 12 patients with premature CAD and 12 age- and sex-matched healthy controls. Six platelet microRNAs were significantly upregulated (miR340*, miR451, miR454*, miR545:9.1. miR615-5p and miR624*) and one miRNA (miR1280) was significantly downregulated in patients with CAD as compared to healthy controls. To validate these results, we measured the expression levels of these candidate miRNAs by qRT-PCR in platelets of individuals from two independent cohorts; validation cohort I consisted of 40 patients with premature CAD and 40 healthy controls and validation cohort II consisted of 27 patients with artery disease and 40 healthy relatives. MiR340* and miR624* were confirmed to be upregulated in patients with CAD as compared to healthy controls in both validation cohorts.

Conclusion/Significance

Two miRNAs in platelets are significantly upregulated in patients with CAD as compared to healthy controls. Whether the two identified miRNAs can be used as biomarkers and whether they are cause or consequence of the disease remains to be elucidated in a larger prospective study.     

成簇pri-miRNAs表达载体的构建及表达验证

目的:旨在建立一个多重表达任意miRNA的方案,实现多个pri-miRNA表达阅读框的串联,用于miRNA簇或者miRNA家族的功能研究。方法:通过改造CRISPR/Cas9系统共表达载体42230,插入EGFP编码序列以及单个pri-miRNA序列,实现单个pri-miRNA表达载体可视化的构建。在单个pri-miRNA表达载体的基础上,通过同源重组的方式,插入下一个pri-miRNA序列,实现多个pri-miRNA表达阅读框的串联表达。结果:以miR29家族的三个miRNA为例,验证了在pri-miRNA表达阅读框串联方面的可行性;在多重的pri-miRNA表达载体中,miRNA成熟体验证结果表明,pri-miR29a和pri-miR29b能够在哺乳动物细胞中被加工成熟,其成熟体明显地过表达。在转染的293A细胞中,miR29家族的靶基因PTEN的表达水平显著降低。结论:所构建的pri-miRNA多重表达方案,能够实现多个外源miRNA在同一细胞中的共表达,对于探索miRNA家族和miRNA簇的拮抗或协同调控功能起到重要的推进作用。     

Differential expression of microRNA in serum fractions and association of Argonaute 1 microRNAs with heart failure

The serum or plasma microRNA (miRNA) molecules have been suggested as diagnostic and prognostic biomarkers, in various pathological conditions. However, these molecules are also found in different serum fractions, such as exosomes and Argonaute (Ago) protein complexes. Ago1 is the predominant Ago protein expressed in heart tissue. The objective of the study was to examine the hypothesis that Ago1‐associated miRNAs may be more relevant to cardiac disease and heart failure compared with the serum. In total, 84 miRNA molecules were screened for their expression in the whole serum, exosomes and Ago1, and Ago2 complexes. Ago1‐bound miR‐222‐3p, miR‐497‐5p and miR‐21‐5p were significantly higher, and let‐7a‐5p was significantly lower in HF patients compared with healthy controls, whereas no such difference was observed for those markers in the serum samples among the groups. A combination of these 4 miRNAs into an Ago1‐HF score provided a ROC curve with an AUC of 1, demonstrating clear discrimination between heart failure patients and healthy individuals. Ago1 fraction might be a better and more specific platform for identifying HF‐related miRNAs compared with the whole serum.     

Expression of multiple artificial microRNAs from a chicken miRNA126-based lentiviral vector

Background

The use of RNAi in both basic and translational research often requires expression of multiple siRNAs from the same vector.

Methods/Principal Findings

We have developed a novel chicken miR126-based artificial miRNA expression system that can express one, two or three miRNAs from a single cassette in a lentiviral vector. We show that each of the miRNAs expressed from the same lentiviral vector is capable of potent inhibition of reporter gene expression in transient transfection and stable integration assays in chicken fibroblast DF-1 cells. Transduction of Vero cells with lentivirus expressing two or three different anti-influenza miRNAs leads to inhibition of influenza virus production. In addition, the chicken miR126-based expression system effectively inhibits reporter gene expression in human, monkey, dog and mouse cells. These results demonstrate that the flanking regions of a single primary miRNA can support processing of three different stem-loops in a single vector.

Conclusions/Significance

This novel design expands the means to express multiple miRNAs from the same vector for potent and effective silencing of target genes and influenza virus.     

Temperature during early development has long-term effects on microRNA expression in Atlantic cod

Background

Environmental temperature has serious implications in life cycle of aquatic ectotherms. Understanding the molecular mechanisms of temperature acclimation and adaptation of marine organisms is of the uttermost importance for ecology, fisheries, and aquaculture, as it allows modeling the effects of global warming on population dynamics. Regulatory molecules are major modulators of acclimation and adaptation; among them, microRNAs (miRNAs) are versatile and substantial contributors to regulatory networks of development and adaptive plasticity. However, their role in thermal plasticity is poorly known. We have asked whether the temperature and its shift during the early ontogeny (embryonic and larval development) affect the miRNA repertoire of Atlantic cod (Gadus morhua), and if thermal experience has long-term consequences in the miRNA profile.

Results

We characterized miRNA during different developmental stages and in juvenile tissues using next generation sequencing. We identified 389 putative miRNA precursor loci, 120 novel precursor miRNAs, and 281 mature miRNAs. Some miRNAs showed stage- or tissue-enriched expression and miRNAs, such as the miR-17 ~ 92 cluster, myomiRs (miR-206), neuromiRs (miR-9, miR-124), miR-130b, and miR-430 showed differential expression in different temperature regimes. Long-term effect of embryonic incubation temperature was revealed on expression of some miRNAs in juvenile pituitary (miR-449), gonad (miR-27c, miR-30c, and miR-200a), and liver (let-7 h, miR-7a, miR-22, miR-34c, miR-132a, miR-192, miR-221, miR-451, miR-2188, and miR-7550), but not in brain. Some of differentially expressed miRNAs in the liver were confirmed using LNA-based rt-qPCR. The effect of temperature on methylation status of selected miRNA promoter regions was mostly inconclusive.

Conclusions

Temperature elevation by several degrees during embryonic and larval developmental stages significantly alters the miRNA profile, both short-term and long-term. Our results suggest that a further rise in seas temperature might affect life history of Atlantic cod.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1503-7) contains supplementary material, which is available to authorized users.