MicroRNA and mRNA expression profiling in metastatic melanoma reveal associations with BRAF mutation and patient prognosis

The role of microRNAs (miRNAs) in melanoma is unclear. We examined global miRNA expression profiles in fresh‐frozen metastatic melanomas in relation to clinical outcome and BRAF mutation, with validation in independent cohorts of tumours and sera. We integrated miRNA and mRNA information from the same samples and elucidated networks associated with outcome and mutation. Associations with prognosis were replicated for miR‐150‐5p, miR‐142‐3p and miR‐142‐5p. Co‐analysis of miRNA and mRNA uncovered a network associated with poor prognosis (PP) that paradoxically favoured expression of miRNAs opposing tumorigenesis. These miRNAs are likely part of an autoregulatory response to oncogenic drivers, rather than drivers themselves. Robust association of miR‐150‐5p and the miR‐142 duplex with good prognosis and earlier stage metastatic melanoma supports their potential as biomarkers. miRNAs overexpressed in association with PP in an autoregulatory fashion will not be suitable therapeutic targets.     

Identification of micro‐RNA networks in end‐stage heart failure because of dilated cardiomyopathy

Micro‐RNAs regulate gene expression by directly binding to the target mRNAs. The goal of the study was to examine the expression profiling of miRNAs in human failing hearts and identify the key miRNAs that regulate molecular signalling networks and thus contribute to this pathological process. The levels of miRNAs and expressed genes were analysed in myocardial biopsy samples from patients with end‐stage heart failure (n = 14) and those from normal heart samples (n = 8). Four networks were built including the Gene regulatory network, Signal‐Network, miRNA‐GO‐Network and miRNA‐Gene‐Network. According to the fold change in the network and probability values in the microarray cohort, RT‐PCR was performed to measure the expression of five of the 72 differentially regulated miRNAs. miR‐340 achieved statistically significant. miR‐340 was identified for the first time in cardiac pathophysiological condition. We overexpressed miR‐340 in cultured neonatal rat cardiomyocytes to identify whether miR‐340 plays a determining role in the progression of heart failure. ANP, BNP and caspase‐3 were significantly elevated in the miR‐340 transfected cells compared with controls (P < 0.05). The cross‐sectional area of overexpressing miR‐340 cardiomyocytes (1952.22 ± 106.59) was greater (P < 0.0001) than controls (1059.99 ± 45.59) documented by Laser Confocal Microscopy. The changes of cellular structure and the volume were statistical significance. Our study provided a comprehensive miRNA expression profiling in the end‐stage heart failure and identified miR‐340 as a key miRNA contributing to the occurrence and progression of heart failure. Our discoveries provide novel therapeutic targets for patients with heart failure.     

Identification of microRNAs and their corresponding targets involved in the susceptibility interaction of wheat response to Puccinia striiformis f. sp. tritici

MicroRNAs (miRNAs) play very important roles in plant defense responses. However, little is known about their roles in the susceptibility interaction between wheat and Puccinia striiformis f. sp. tritici (Pst). In this study, two miRNA libraries were constructed from the leaves of the cultivar Xingzi 9104 inoculated with the virulent Pst race CYR32 and sterile water, respectively. A total of 1316 miRNA candidates, including 173 known miRNAs that were generated from 98 pre‐miRNAs, were obtained. The remaining 1143 miRNA candidates included 145 conserved and 998 wheat‐specific miRNAs that were generated from 87 and 1088 pre‐miRNAs, respectively. The 173 known and 145 conserved miRNAs were sub‐classified into 63 miRNA families. The target genes of wheat miRNAs were also confirmed using degradome sequencing technology. Most of the annotated target genes were related to signal transduction or energy metabolism. Additionally, we found that miRNAs and their target genes form complicated regulation networks. The expression profiles of miRNAs and their corresponding target genes were further analyzed by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the results indicate that some miRNAs are involved in the compatible wheat‐Pst susceptibility interaction. Importantly, tae‐miR1432 was highly expressed when wheat was challenged with CYR32, and the corresponding target gene, predicted to be a calcium ion‐binding protein, also exhibited upregulated expression but a divergent expression trend. PC‐3P‐7484, a specific wheat miRNA, was highly expressed in the wheat response to Pst infection, while the expression of the corresponding target gene ubiquillin was dramatically downregulated. These data provide the foundation for evaluating the important regulatory roles of miRNAs in wheat‐Pst susceptibility interaction.     

Naive and primed murine pluripotent stem cells have distinct miRNA expression profiles

Over the last years, the microRNA (miRNA) pathway has emerged as a key component of the regulatory network of pluripotency. Although clearly distinct states of pluripotency have been described in vivo and ex vivo, differences in miRNA expression profiles associated with the developmental modulation of pluripotency have not been extensively studied so far. Here, we performed deep sequencing to profile miRNA expression in naive (embryonic stem cell [ESC]) and primed (epiblast stem cell [EpiSC]) pluripotent stem cells derived from mouse embryos of identical genetic background. We developed a graphical representation method allowing the rapid identification of miRNAs with an atypical profile including mirtrons, a small nucleolar RNA (snoRNA)-derived miRNA, and miRNAs whose biogenesis may differ between ESC and EpiSC. Comparison of mature miRNA profiles revealed that ESCs and EpiSCs exhibit very different miRNA signatures with one third of miRNAs being differentially expressed between the two cell types. Notably, differential expression of several clusters, including miR290-295, miR17-92, miR302/367, and a large repetitive cluster on chromosome 2, was observed. Our analysis also showed that differentiation priming of EpiSC compared to ESC is evidenced by changes in miRNA expression. These dynamic changes in miRNAs signature are likely to reflect both redundant and specific roles of miRNAs in the fine-tuning of pluripotency during development.     

Profiling highly conserved microrna expression in recombinant IgG‐producing and parental Chinese hamster ovary cells

MicroRNAs (miRNAs) play important roles in global gene regulation. Researchers in recombinant protein production have proposed miRNAs as biomarkers and cell engineering targets. However, miRNA expression remains understudied in Chinese Hamster Ovary cells, one of the most commonly used host cell systems for therapeutic protein production. To profile highly conserved miRNA expression, we used the miRCURY? miRNA array for screening miRNAs in CHO cells. The selection criteria for further miRNA profiling included positive hybridization signals and experimentally validated predicted regulatory targets. On the basis of screening, we selected 16 miRNAs for quantitative RT‐PCR profiling. We profiled miR expression in parental CHO DG44 and CHO K1 cell lines as well as four recombinant DG44‐derived CHO lines producing a recombinant human IgG. We observed that miR‐221 and miR‐222 were significantly downregulated in all IgG‐producing cell lines when compared with parental DG44, whereas miR‐125b was significantly downregulated in one IgG‐producing line. In another IgG‐producing line, miR‐19a was significantly upregulated. miRNA expression was also profiled in two of these lines that were amplified by stepwise increase of methotrexate. In both amplified cell lines, let‐7b and miR‐221 were significantly downregulated. In parental CHO K1, let‐7b, miR‐15b, and miR‐17 were significantly downregulated when compared with DG44. The results reported here are the first steps toward profiling highly conserved miRNAs and studying the clonal difference in miRNA expression in CHO cells and may shed light on using miRNAs in cell engineering. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Genome‐wide identification of urinary cell‐free microRNAs for non‐invasive detection of bladder cancer

Urinary microRNAs (miRNAs) are emerging as clinically useful tool for early and non‐invasive detection of various types of cancer including bladder cancer (BCA). In this study, 205 patients with BCA and 99 healthy controls were prospectively enrolled. Expression profiles of urinary miRNAs were obtained using Affymetrix miRNA microarrays (2578 miRNAs) and candidate miRNAs further validated in independent cohorts using qRT‐PCR. Whole‐genome profiling identified 76 miRNAs with significantly different concentrations in urine of BCA compared to controls (P < 0.01). In the training and independent validation phase of the study, miR‐31‐5p, miR‐93‐5p and miR‐191‐5p were confirmed to have significantly higher levels in urine of patients with BCA in comparison with controls (P < 0.01). We further established 2‐miRNA‐based urinary DxScore (miR‐93‐5p, miR‐31‐5p) enabling sensitive BCA detection with AUC being 0.84 and 0.81 in the training and validation phase, respectively. Moreover, DxScore significantly differed in the various histopathological subgroups of BCA and decreased post‐operatively. In conclusion, we identified and independently validated cell‐free urinary miRNAs as promising biomarkers enabling non‐invasive detection of BCA.     

Serum miRNAs are potential biomarkers for the detection of disc degeneration,among which miR‐26a‐5p suppresses Smad1 to regulate disc homeostasis

Disc degeneration is a common clinical condition in which damaged discs cause chronic pain; however, a laboratory diagnosis method for its detection is not available. As circulating miRNAs have potential as biomarkers, their application in disc degeneration has not been explored. Here, we prepared serum miRNAs from a mouse disc degeneration model and performed miRNA‐Seq and quantitative PCR to characterize disc degeneration–associated miRNAs. We identified three miRNAs, including miR‐26a‐5p, miR‐122‐5p and miR‐215‐5p, undergoing perturbation during the pathogenesis of disc degeneration. Specifically, the levels of miR‐26a‐5p in the serum demonstrated steady increases in the model of disc degeneration, compared with those in the pre‐injury samples of younger age or compared with normal controls of the same age but without disc degeneration, whereas the miRNAs miR‐122‐5p and miR‐215‐5p exhibited lower expression in post‐injury samples than in their counterparts without the surgery. Moreover, we found that miR‐26a‐5p targets Smad1 expression, and Smad1 negatively regulates Vegfa expression in disc cells, and thus, miR‐26a‐5p promotes disc degeneration. In summary, we established a method that consistently profiles circulating miRNAs and identified multiple miRNAs as promising biomarkers for disc degeneration, among which miR‐26a‐5p enhances VEGF expression during disc degeneration through targeting Smad1 signalling.     

胡杨无性系幼苗响应盐胁迫的miRNA表达差异研究

植物miRNA在调控基因表达、细胞周期、生物体发育、抗逆等方面起重要作用。为研究胡杨(Populus euphratica Oliv.)的耐盐机制,以1年生胡杨无性系幼苗为材料,构建具有空间代表性的盐胁迫胡杨cDNA文库,利用二代测序技术测定NaCl胁迫下和正常培养条件下胡杨叶和根miRNA表达情况。结果表明,不同的miRNA之间表达量存在明显差异,表达丰度最高的miRNA有miR156、miR157、miR165、miR166和miR167等,合计占总表达量的90%以上。胡杨根部存在特异表达的miRNA,在整个耐盐调控机制中发挥着生理调节、分子调控和信号传导等极为重要的作用。盐处理样品中发现大量响应盐胁迫的miRNA,对这些转录因子进行靶基因预测和注释后,发现很多盐胁迫响应的miRNA与NAC和SPL等重要转录因子家族相关,与前人的结论一致,另外还发现许多miRNA的调控对象是ATP酶和激素响应因子。     

Altered microRNA expression in bovine skeletal muscle with age

Age‐dependent decline in skeletal muscle function leads to several inherited and acquired muscular disorders in elderly individuals. The levels of microRNAs (miRNAs) could be altered during muscle maintenance and repair. We therefore performed a comprehensive investigation for miRNAs from five different periods of bovine skeletal muscle development using next‐generation small RNA sequencing. In total, 511 miRNAs, including one putatively novel miRNA, were identified. Thirty‐six miRNAs were differentially expressed between prenatal and postnatal stages of muscle development including several myomiRs (miR‐1, miR‐206 and let‐7 families). Compared with miRNA expression between different muscle tissues, 14 miRNAs were up‐regulated and 22 miRNAs were down‐regulated in the muscle of postnatal stage. In addition, a novel miRNA was predicted and submitted to the miRBase database as bta‐mir‐10020. A dual luciferase reporter assay was used to demonstrate that bta‐mir‐10020 directly targeted the 3′‐UTR of the bovine ANGPT1 gene. The overexpression of bta‐mir‐10020 significantly decreased the DsRed fluorescence in the wild‐type expression cassette compared to the mutant type. Using three computational approaches – miranda , pita and rnahybrid – these differentially expressed miRNAs were also predicted to target 3609 bovine genes. Disease and biological function analyses and the KEGG pathway analysis revealed that these targets were statistically enriched in functionality for muscle growth and disease. Our miRNA expression analysis findings from different states of muscle development and aging significantly expand the repertoire of bovine miRNAs now shown to be expressed in muscle and could contribute to further studies on growth and developmental disorders in this tissue type.