MicroRNA - Volume 2, Issue 1, 2013

Human papillomaviruses (HPVs) are associated with the pathogenesis of a variety of human cancers, including cervical and oropharyngeal cancers. The HPV E6 and E7 oncogenes are usually expressed to high levels in these cancers. Previous studies have shown dysregulation of cellular microRNAs (miRNAs) in HPV-positive cell lines and cancer tissues and recent studies have identified a few miRNAs whose levels are altered in the presence of the viral E6 and E7 proteins. In order to identify all the cellular miRNAs whose expression may be affected by these oncoproteins, we carried out microarray analysis using human foreskin keratinocytes (HFKs) expressing either or both of these two proteins. These studies showed that 90 and 60 miRNAs were dysregulated in the presence of the E6 or the E7 protein, respectively. Of these, 43 miRNAs were similarly affected in HFK-E6 and/or HFK-E7 when compared to control cells. The joint expression of E6 and E7 proteins in HFKs caused changes in the levels of 64 miRNAs, of which 24 were similarly affected in HFK-E6 and/or HFK-E7 cells relative to controls. The microarray experiments were validated by quantitative real-time RT-PCR analysis of several differentially expressed miRNAs. Several miRNAs dysregulated by the E6 and/or E7 proteins are known to be altered in a variety of human cancers. Furthermore, previously known cellular targets of these miRNAs are involved in processes such as cell cycle regulation, apoptosis, cell-cell adhesion, cell mobility and proliferation, and alterations in their levels may contribute to HPV-associated carcinogenesis. Taken together, the results of our studies suggest that high risk HPV E6 and E7 proteins share the ability to regulate a subset of cellular miRNAs.

MicroRNAs (miRNAs) have been found to be stable in the circulation of cancer patients raising their potential as circulating biomarkers of disease. The specific source and role, however, of miRNAs in the circulation is unknown and requires elucidation to determine their true potential. In this study, along with primary tissue explants and primary stromal cells, three breast cancer cell lines were employed, including T47D, MDA-MB-231 and SK-BR-3. Tissue explants were harvested in theatre, with informed patient consent, and included tumour, tumour associated normal, and diseased lymph node samples. Cell-conditioned media containing all factors secreted by the cells were harvested. MiRNAs were extracted from samples using 5 different extraction techniques including the blood protocol, RNeasy® (Qiagen), miRNeasy®mini kit (Qiagen), mirVana™ isolation kit (Ambion) and RNAqueous® kit (Ambion). MiRNAs were successfully isolated from all media samples collected from cell lines, primary cells and fresh tissue explants. However, there was remarkable variation in yield depending on the extraction method used. Aliquots of the same samples were extracted, revealing the two column extraction protocol of the mirVana® miRNA isolation kit to be the most suitable approach. A range of miRNAs, including miR-16, miR-195, miR-497 and miR-10b, were successfully amplified. While miR-16 and miR-195 were detected in media from both cell lines and tissue explants, miR-497 and miR-10b were only detected in secretions from whole tissue explants. The ability to achieve reliable and reproducible miRNA yields from cell-conditioned media is vital for the successful amplification of miRNAs by RQ-PCR.

MicroRNAs (miRNAs), which mainly inhibit protein expression by targeting the 3’UTR (untranslated region) of mRNAs, are known to play various roles in the pathogenesis of many different types of diseases. Specifically, in bone diseases, recent emphasis has been placed on the involvement of miRNAs in the differentiation and proliferation of bone and cartilage cells, particularly with regards to how these mechanisms contribute to bone homeostasis. In this review, we summarize miRNAs that are important in the differentiation and proliferation of bone cells, and specific miRNAs associated with bone diseases, such as osteoporosis, osteoarthritis and rheumatoid arthritis. This review also provides the perspective that miRNA studies will identify not only new mechanisms in basic bone research, but also potential novel diagnostic biomarkers and drug targets for bone diseases.

Microvascular adaptation to metabolic stress is important in the maintenance of tissue homeostasis. Nowhere is this more important than in the central nervous system (CNS) where the cellular constituents of the neurovascularture including endothelial cells, pericytes and some astroglia must make fine-tuned autoregulatory modulations that maintain the delicate balance between oxygen availability and metabolic demand. miRNAs have been reported to play an important regulatory role in many cellular functions including cell differentiation, growth and proliferation, lineage determination, and metabolism. In this study, we investigated the possible role of miRNAs in the CNS capillary pericyte response to hypoxic stress. Micro-array analysis was used to examine the expression of 388 rat miRNAs in primary rat cortical pericytes with and without exposure to low oxygen (1%) after 24 or 48 hr. Pericytes subjected to hypoxia showed 27 miRNAs that were higher than control and 31 that were lower. Validation and quantification was performed by Real Time RT-PCR on pericytes subjected to 2 hr, 24 hr, or 48 hr of hypoxia. Hypoxia induced changes included physiological pathways governing the stress response, angiogenesis, migration and cell cycle regulation. miRNAs associated with HIF-1α (miR-322[1], miR-199a [2]), TGF-β1 (miR-140[3], miR-145[4], miR-376b-3p[5]) and VEGF (miR-126a[6], miR-297[7], miR-16[8], miR-17-5p[9]) were differentially regulated. Systematic and integrative analysis of possible gene targets analyzed by DAVID bioinformatics resource (http://david.abcc.ncifcrf.gov) and MetaSearch 2.0 (GeneGo) for some of these miRNAs was conducted to determine possible gene targets and pathways that may be affected by the post-transcriptional changes after hypoxic insult.

Apomixis refers to plant asexual reproduction through seeds that give rise to progeny which are genotypically identical to the maternal parent. It has evolved from many different sexual taxa although the underlying genetic factors remain unknown. Previous analyses of the over-representation of transcription factors, in a comparison of microdissected ovules from apomictic and sexual Boechera, showed that many transcription factor mRNAs possessed microRNA (miRNAs) binding sites, thus pointing to miRNAs as potentially important factors that may be involved in the regulatory switch from sexual to apomictic reproduction. A microarray-based approach was used to identify (1) 673 microsatellitelike small RNAs (misRNAs) containing predominantly 2-7 repeats of (GAA)n/(CUU)n, (GCA)n/(CGU)n, (GGA)n/(CCU)n, (GGU)n/(CCA)n and (UGA)n/(ACU)n, and (2) 166 more typical non-repeat small RNAs. In total, 87 small RNAs were found to be located in cDNAs that could fold into stem-loop structures and thus represent miRNA molecules. In addition, 109 Boechera small RNAs including both misRNAs and non-repeat small RNAs, showed significant homology to 407 Arabidopsis thaliana small RNAs including the A. thaliana pollen-specific ath-miR5021. This indicates that only a fraction of the identified small RNAs are unique to Boechera. Ten small RNAs were validated using a Northern blot assay on flower and leaf tissues, eight of which showed flower-specific expression with varying abundance. The potential binding sites of many of the misRNAs and non-repeat small RNAs occur predominantly in exonic regions. This feature coupled with their flower-specific pattern of expression is suggestive of their probable role in post-transcriptional gene regulation. We propose that quantitative variation for misRNA target binding (and hence post-transcriptional gene regulation) could arise via microsatellite length polymorphisms occurring either in misRNA precursors or in their gene targets.

MicroRNAs (miRNAs), a class of small, non-coding RNA molecules with gene regulatory functions, have emerged to play a critical role in the pathogenesis of a variety of diseases. Current technological advances allow accurate, high throughput profiling of miRNA abundance in different tissues. More recently, extracellular, circulating miRNAs have begun to be demonstrated as highly stable, blood-based biomarkers for diseases. Understanding the interactions between circulating miRNAs and clinical phenotypes can enhance our knowledge of complex diseases and traits. On the other hand, given the advantages of utilizing blood-based biomarkers (e.g., convenience in collecting samples), circulating miRNAs as biomarkers may improve both disease diagnosis and management. Particularly, we reviewed recent progress in identifying circulating miRNAs as biomarkers for several common inflammatory diseases including asthma, inflammatory bowel disease, and rheumatoid arthritis. Current studies showed a promising future of using circulating miRNAs in the care of inflammatory diseases.

Micro RNAs (miRNAs) are short non-coding RNAs of 20-24nt in length mediating RNA silencing, a eukaryotic, sequence-specific repressive gene regulation mechanism. In plants, miRNAs have a pivotal role during fundamental processes such as development, maintenance of genome integrity and abiotic stress responses. They originate from MIRNA genes that are transcribed by RNA polymerase II; MIRNA transcripts form imperfect fold-back structures that are further processed to miRNA duplexes. In Arabidopsis, over 180 MIRNA loci have been identified. Recent evidence shows that miRNAs are substantially implicated in regulating plant immunity. Pathogen attack triggers massive changes in the miRNA transcriptome; many of the altered miRNAs participate in controlling plant hormone pathways. Moreover, microorganisms are known to manipulate silencing pathways to counteract miRNA-mediated defenses. Thus far, miRNAs are believed to likely function as cardinal players in the concert of broad-spectrum disease resistance. Here, we summarize the highlights and latest findings of miRNAs as molecular regulators during plant-microbe interactions.