siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.     

Liquid biopsy: miRNA as a potential biomarker in oral cancer

Oral cancer is one of the leading cancers in South-Asian countries. Despite the easy access of the oral cavity, the detection and five year survival rates of OSCC patients are dismal. Identification of non-invasive biomarkers to determine the progression and recurrence of OSCC could be of immense help to patients. Recent studies on oral cancer suggest the importance of non-invasive biomarker development. Micro-RNAs (miRNAs) are one of the important components of the cell-free nucleic acids available in different body fluids. Here, we have reviewed the current understanding of circulating miRNAs as non-invasive biomarkers in different body fluids of oral cancer patients. A number of circulating miRNAs are found to be common in the body fluids of OSCC patients, while many of these are study specific, the possible sources of this variability could be due to differences in sample processing, assay procedure, clinical stage of the disease, oral habit and environmental factors. The prognostic and therapeutic significance of these circulating miRNAs are suggested by several studies. Mir-371, mir-150, mir-21 and mir-7d were found to be potential prognostic markers, while mir-134, mir-146a, mir-338 and mir-371 were associated with metastases. The prognostic markers, mir-21 and mir-7d were also found to be significantly correlated with resistance to chemotherapy, while mir-375, mir-196 and mir-125b were significantly correlated with sensitivity to radiotherapy. Despite the promising roles of circulating miRNAs, challenges still remain in unravelling the exact regulation of these miRNAs before using them for targeted therapy.     

Distribution of miRNA genes in the pig genome

Background

Recent completion of swine genome may simplify the production of swine as a large biomedical model. Here we studied sequence and location of known swine miRNA genes, key regulators of protein-coding genes at the level of RNA, and compared them to human and mouse data to prioritize future molecular studies.

Results

Distribution of miRNA genes in pig genome shows no particular relation to different genomic features including protein coding genes - proportions of miRNA genes in intergenic regions, introns and exons roughly agree with the size of these regions in the pig genome. Our analyses indicate that host genes harbouring intragenic miRNAs are longer from other protein-coding genes, however, no important GO enrichment was found. Swine mature miRNAs show high sequence similarity to their human and mouse orthologues. Location of miRNA genes relative to protein-coding genes is also similar among studied species, however, there are differences in the precise position in particular intergenic regions and within particular hosts. The most prominent difference between pig and human miRNAs is a large group of pig-specific sequences (53% of swine miRNAs). We found no evidence that this group of evolutionary new pig miRNAs is different from old miRNAs genes with respect to genomic location except that they are less likely to be clustered.

Conclusions

There are differences in precise location of orthologues miRNA genes in particular intergenic regions and within particular hosts, and their meaning for coexpression with protein-coding genes deserves experimental studies. Functional studies of a large group of pig-specific sequences in future may reveal limits of the pig as a model organism to study human gene expression.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0166-3) contains supplementary material, which is available to authorized users.     

miRConnect: identifying effector genes of miRNAs and miRNA families in cancer cells

micro(mi)RNAs are small non-coding RNAs that negatively regulate expression of most mRNAs. They are powerful regulators of various differentiation stages, and the expression of genes that either negatively or positively correlate with expressed miRNAs is expected to hold information on the biological state of the cell and, hence, of the function of the expressed miRNAs. We have compared the large amount of available gene array data on the steady state system of the NCI60 cell lines to two different data sets containing information on the expression of 583 individual miRNAs. In addition, we have generated custom data sets containing expression information of 54 miRNA families sharing the same seed match. We have developed a novel strategy for correlating miRNAs with individual genes based on a summed Pearson Correlation Coefficient (sPCC) that mimics an in silico titration experiment. By focusing on the genes that correlate with the expression of miRNAs without necessarily being direct targets of miRNAs, we have clustered miRNAs into different functional groups. This has resulted in the identification of three novel miRNAs that are linked to the epithelial-to-mesenchymal transition (EMT) in addition to the known EMT regulators of the miR-200 miRNA family. In addition, an analysis of gene signatures associated with EMT, c-MYC activity, and ribosomal protein gene expression allowed us to assign different activities to each of the functional clusters of miRNAs. All correlation data are available via a web interface that allows investigators to identify genes whose expression correlates with the expression of single miRNAs or entire miRNA families. miRConnect.org will aid in identifying pathways regulated by miRNAs without requiring specific knowledge of miRNA targets.     

Binding sites for peptidoglycan on mouse lymphocytes

Binding of peptidoglycan (PG), a B-cell mitogen and polyclonal activator, to mouse lymphocytes was studied using rosetting with PG-sensitized erythrocytes and a direct binding assay with 125I-labeled PG. Thirty-four percent of splenic lymphocytes formed PG rosettes, 62% of which were inhibited by preincubation of lymphocytes with free PG. Less than 1 or 3% of spleen cells formed rosettes with uncoated or albumin-coated red cells. The formation of rosettes was not inhibited by 0.1% azide and was not dependent on the presence of complement or immunoglobulins. The 125I-PG bound both specifically and nonspecifically to the lymphocytes. The binding was completed within 15-20 min, was proportional to the cell concentration, and was not inhibited by 0.1% azide or treatment of lymphocytes with formalin. The cells had one set of specific binding sites of low affinity (KD = 1.2-4.6 X 10(-7) M +/- 9% SE, based on competitive) experiments. The binding, however, was complex, probably involving interaction of multiple binding sites on PG with the cell surface. The EC50 (920 micrograms/ml) was similar to the optimal lymphocyte-activating concentration of PG (400-1000 micrograms/ml). The binding correlated with the ability of different PG preparations to stimulate lymphocytes, since only high Mr PG (not low Mr PG preparations, muramyl dipeptide (MDP), or PG pentapeptide) had the ability to specifically bind to lymphocytes, to compete with PG binding, and to stimulate lymphocytes. Also, low Mr PG preparations, MDP, or PG pentapeptide did not inhibit the mitogenic stimulation of lymphocytes by high Mr PG. These results indicate the presence of specific binding sites for PG on the surface of murine lymphocytes and suggest that the binding of PG to these binding sites is involved in lymphocyte activation by PG.     

Binding sites for horseradish peroxidase on the cell surface

Summary The cytochemical reaction for surface-bound horseradish peroxidase (HRP) on cultured HeLa cells, GH₃ cells, and isolated rat liver cells was suppressed by 30 M monosialoganglioside, by 30 M trisialoganglioside, or by 5 mM CMP-neurminic acid. The reaction was also suppressed by 10 mM chitotriose or by 10 mM UDP-galactose, a galactose acceptor and donor, respectively, for galactosyltransferase. The addition of 2 mM Mn²⁺ to the incubation medium with HRP suppressed the reaction for surfacebound HRP, and the addition of 10–20 mM Ca²⁺ intensified the reaction. The addition of 2 mM Zn²⁺ caused less inhibition than that of 2 mM Mn²⁺, and the addition of 2 mM Co²⁺ caused either a slight inhibition, or no inhibition. These observations support the hypothesis that HRP may be bound to a glycosyltransferase at the cell surface.     

microRNA与食管癌关系的研究进展

肿瘤的发生、发展是多基因共同参与的多步骤的复杂过程,包括癌基因的异常激活和抑癌基因的失活。MicroRNAs(miRNAs)是一组真核细胞内源性产生的单链小RNA分子,研究发现miRNAs的表达异常与人类肿瘤发生有着密切的关系。食管癌是我国常见的恶性肿瘤之一,近来miRNA与食管癌的相关研究也日渐报道,因此本文就此作一综述。     

OrCGDB: a database of genes involved in oral cancer

The Oral Cancer Gene Database (OrCGDB; http://www.tumor-gene. org/Oral/oral.html) was developed to provide the biomedical community with easy access to the latest information on the genes involved in oral cancer. The information is stored in a relational database and accessed through a WWW interface. The OrCGDB is organized by gene name, which is linked to information describing properties of the gene. This information is stored as a collection of findings ('facts') that are entered by the database curator in a semi-structured format from information in primary publications using a WWW interface. These facts include causes of oncogenic activation, chromosomal localization of the gene, mutations associated with the gene, the biochemical identity and activity of the gene product, synonyms for the gene name and a variety of clinical information. Each fact is associated with a MEDLINE citation. The user can search the OrCGDB by gene name or by entering a textword. The OrCGDB is part of a larger WWW-based tumor gene database and represents a new approach to catalog and display the research literature.     

The use of miRNA microarrays for the analysis of cancer samples with global miRNA decrease

Recent studies have established that mutations or deletions in microRNA (miRNA) processing enzymes resulting in a global decrease of miRNA expression are frequent across cancers and can be associated with a poorer prognosis. While very popular in miRNA profiling studies, it remains unclear whether miRNA microarrays are suited or not to accurately detecting global miRNA decreases seen in cancers. In this work, we analyzed the miRNA profiles of samples with global miRNA decreases using Affymetrix miRNA microarrays following the inducible genetic deletion of Dicer1. Surprisingly, up to a third of deregulated miRNAs identified upon Dicer1 depletion were found to be up-regulated following standard robust multichip average (RMA) background correction and quantile normalization, indicative of normalization bias. Our comparisons of five preprocess steps performed at the probe level demonstrated that the use of cyclic loess relying on non-miRNA small RNAs present on the Affymetrix platform significantly improved specificity and sensitivity of detection of decreased miRNAs. These findings were validated in samples from patients with prostate cancer, where conjugation of robust normal-exponential background correction with cyclic loess normalization and array weights correctly identified the greatest number of decreased miRNAs, and the lowest amount of false-positive up-regulated miRNAs. These findings highlight the importance of miRNA microarray normalization for the detection of miRNAs that are truly differentially expressed and suggest that the use of cyclic loess based on non-miRNA small RNAs can help to improve the sensitivity and specificity of miRNA profiling in cancer samples with global miRNA decrease.     

Current tools for the identification of miRNA genes and their targets

The discovery of microRNAs (miRNAs), almost 10 years ago, changed dramatically our perspective on eukaryotic gene expression regulation. However, the broad and important functions of these regulators are only now becoming apparent. The expansion of our catalogue of miRNA genes and the identification of the genes they regulate owe much to the development of sophisticated computational tools that have helped either to focus or interpret experimental assays. In this article, we review the methods for miRNA gene finding and target identification that have been proposed in the last few years. We identify some problems that current approaches have not yet been able to overcome and we offer some perspectives on the next generation of computational methods.     

Two populations of TSPO binding sites in oral cancer SCC-15 cells

Oral cancer mortality and morbidity rates remain high. The main inducer of oral cancer is cigarette smoke (CS). Translocator protein 18 kDa (TSPO) was shown to play a role in carcinogenesis. We characterized TSPO binding sites in human oral cancer cell line SCC-15 and examined effect of CS on TSPO binding. We exposed SCC-15 human squamous cells to cigarette smoke. [³H]PK 11195 binding results were assessed in cells confluent for one day. To characterize the number of population sites, a custom written Matlab program compared Pearson linear correlation coefficients between all points in the Scatchard plot. Using [³H]PK 11195 as a radio ligand, we found that TSPO binding sites are not uniform, but separated into two sub-populations, one with high affinity (respective Kd and Bmax values of 1.40±0.08 nM and 1586±48 fmol/mg protein), another with lower affinity (respective Kd and Bmax values of 61±5 nM and 26260±1050 fmol/mg protein). We demonstrate rapid decrease in TSPO binding to the high affinity site induced by exposure to CS; specifically, significant 36% decrease in binding after 30 min CS exposure (p<0.05), and 69% decrease after 2 h CS exposure (p<0.05). Association between TSPO and CS exposure may contribute to understanding the underlying mechanism of oral carcinogenesis.     

Binding sites of calmodulin and actin on the brain spectrin, calspectin

We used rotary-shadowing electron microscopy to map the calmodulin-and actin-binding sites on the brain spectrin, calspectin (or fodrin). Calspectin dimers appeared as rods 110 nm long and joined in a head-to- head manner to form tetramers 220 nm long. We determined calmodulin- binding sites by a ferritin-labeling method combined with biotin-avidin complex formation. Ferritin particles were found to attach to the head parts of calspectin dimers at a position 10-20 nm from the top of the head. The number of the calmodulin-binding sites seemed to be only one for each dimer and two for each tetramer. In contrast, the actin- binding sites were localized at the tail ends of the calspectin molecules. The tetramers attached to muscle F-actin with their tail ends and often cross-linked adjacent filaments. The results are discussed in view of the analogy to the erythrocyte spectrin.