Genomic instability and mouse microRNAs

Tumor progression is the continual selection of variant subpopulations of malignant cells that have acquired increasing levels of genetic instability (Nowell Science 1976, 194, 23–28). This instability is manifested as chromosomal aneuploidy or translocations, viral integration or somatic mutations that typically affect the expression of a gene (oncogene) that is especially damaging to the proper function of a cell. With the recent discovery of non-coding RNAs such as microRNAs (miRNAs), the concept that a target of genetic instability must be a protein-encoding gene is no longer tenable. Over the years, we have conducted several studies comparing the location of miRNA genes to positions of genetic instability, principally retroviral integration sites and chromosomal translocations in the mouse as a means of identifying miRNAs of importance in carcinogenesis. In this current study, we have used the most recent annotation of the mouse miRome (miRBase, release 16.0), and several datasets reporting the sites of integration of different retroviral vectors in a variety of mouse strains and mouse models of cancer, including for the first time a model that shows a propensity to form solid tumors, as a means to further identify or define, candidate oncogenic miRNAs. Several miRNA genes and miRNA gene clusters stand out as interesting new candidate oncogenes due to their close proximity to common retroviral integration sites including miR-29a/b/c and miR106a~363. We also discussed some recently identified miRNAs including miR-1965, miR-1900, miR-1945, miR-1931, miR-1894, and miR-1936 that are close to common retroviral integration sites and are therefore likely to have some role in cell homeostasis.     

Genomic sequence analysis of EGFR regulation by microRNAs in lung cancer

Lung cancer is known as the top cancer killer in most developed countries. Epidermal growth factor receptor (EGFR) is frequently found to be activated by mutation or amplification in lung cancer. MicroRNA (miRNA) is a new class of small molecules that has emerged as important markers of lung cancer development and therapeutic target. There are queries on which miRNAs can regulate EGFR and it is important to predict the candidate miRNAs that target EGFR by bioinformatics and to investigate on the availability of these candidate miRNA regulators in lung cancer. Systematic and rigorous searches for miRNAs targeting EGFR were performed on 10 representative databases. The identified miRNAs that target EGFR were formulated into a conditional regulation matrix and then hierarchical clustering algorithm was applied for the analysis. The systematic search came up with 138 miRNAs that potentially target EGFR. Among them, 11 miRNAs including miR-7 and miR-128b were confirmed by published experimental data or literatures. There were 14 candidate miRNAs predicted by at least 3 prediction pipelines in this study which have never been previously reported to target EGFR. Further studies of these novel identified miRNAs may provide insight on the regulation of EGFR in lung cancer. To the best of our knowledge, this is the first bioinformatic study applying genomic sequence analysis for the prediction of miRNAs that target EGFR in lung cancer. This new strategy that integrates computational and published data approaches provides a fast and effective prediction of miRNAs in specific target genes involved in various diseases.     

The changes of miRNA expression in rat hippocampus following chronic lead exposure

miRNAs have been found to contribute to normal brain functions, nervous system diseases, as well as neurotoxicities induced by external agents. However, whether they are involved in lead-induced neurotoxicities is still not clear. To identify that, a lead-induced chronic neurotoxicity model of rats was built. Both miRNA microarray analysis and qRT-PCR were performed to determine the change of miRNA expression in hippocampus. Then 3 bioinformatics databases were used to analyze the relative target genes of these miRNA, which were further confirmed by qRT-PCR and Western blot. In the present study, lead exposure resulted in the changed expression of 7 miRNAs: miR-204, miR-211, miR-448, miR-449a, miR-34b, and miR-34c were greatly up-regulated while miR-494 was greatly down-regulated. Bioinformatics analysis results showed that the target genes of 6 up-regulated miRNAs were related to neural injury and neurodegeration, axon and synapse function, neural development and regeneration. Correspondingly, the expression levels of mature mRNAs and proteins of three target genes (Bcl-2, Itpr1, and Map2k1) were greatly repressed, verifying the results of bioinformatics analysis. Taken together, our results showed that the expression of several miRNAs reported to be associated with neurophysiological pathways and neurodegenerative diseases changed in rat hippocampus following chronic lead exposure. These miRNAs may play important roles in lead-induced neurotoxicity.     

miRNA: small molecules as potential novel biomarkers in cancer

Four different types of small RNAs functionally associated with gene silencing have been discovered in animals including small interfering RNAs (siRNAs), microRNAs (miRNAs), and Piwi-interacting RNAs (piRNAs). Experimental evidence suggests that miRNAs regulate the expression of more than 30% of protein-coding genes. These molecules can also act as oncogenes or tumor suppressors. Expression profiling has revealed characteristic miRNA signatures not only in human cancers but also in serum and blood cells of cancer patients. Numerous human miRNA genes map to chromosomal regions which are susceptible to amplification, deletion or translocation in the process of tumor development. Despite the pivotal role of miRNA in cancer precise mechanisms of action are yet to be elucidated. This review is focused on recent findings related to the emerging field of miRNA serving as novel potential biomarkers in cancer diagnosis, prognosis and possibly, therapies.     

Targeted Stage-Specific Inflammatory microRNA Profiling in Urine During Disease Progression in Experimental Autoimmune Encephalomyelitis: Markers of Disease Progression and Drug Response

Recently, microRNAs (miRNAs) have been implicated in regulating neuroinflammatory and demyelinative responses in multiple sclerosis (MS) and its mouse model of experimental autoimmune encephalomyelitis (EAE). miRNAs have also been studied as biomarkers of disease pathology and drug-response in MS. However, no complete miRNA profiling at various stages of EAE disease has been examined, especially in the urine. We carried out a systematic analysis of miRNAs in the urine exosomes as well as in the plasma and spinal cord at pre-onset, onset and peak stages of EAE established in the chronic B6 mice model. For the first time, we provide evidence that urine exosomes can be a specific and sensitive source of miRNA biomarkers for all 3 stages of EAE disease. In a significant observation, we observed that miR-155-5p expression increased in urine exosomes, plasma and spinal cord 6 days before the onset of disease, suggesting its early involvement in the pathology of EAE disease. We also analyzed the effect of Glatiramer acetate (GA; copaxone) treatment, an approved treatment for MS patients, in modulating miRNA expression at the peak of EAE disease. We identified miR-155-5p, miR-27a-3p, miR-9-5p and miR-350-5p as putative GA-treatment responsive miRNA biomarkers. Since, EAE is a mainly CD4 cells mediated disease, we also examined the above set of miRNAs and found to be significantly altered in T cells polarized to Th1 and Th17 phenotype, similar to urine exosomes. Thus, urine exosome miRNAs hold the potential to be defined as novel accessible stage-specific biomarkers of EAE (MS) disease as well as treatment response.     

Evaluation of single nucleotide polymorphisms in the miR-183–96–182 cluster in adulthood attention-deficit and hyperactivity disorder (ADHD) and substance use disorders (SUDs)

Attention deficit-hyperactivity disorder (ADHD) is a neuropsychiatric disorder characterized by inappropriate and impaired levels of hyperactivity, impulsivity and inattention. Around 75% of adults with ADHD show comorbidity with other psychiatric disorders such as disruptive behavior disorders or substance use disorders (SUDs). Recently, there has been growing interest in studying the role of microRNAs (miRNAs) in the susceptibility to complex disorders. Interestingly, converging evidence suggests that single nucleotide polymorphisms (SNPs) within miRNAs or miRNA target sites may modulate the miRNA-mediated regulation of gene expression through the alteration of the miRNA maturation, structure or expression pattern as well as the silencing mechanisms of target genes. Genetic studies and animal models support the involvement of the serotonin receptor (HTR1B) in ADHD. We evaluated the contribution of one SNP in the miR-96 target site at HTR1B and eight tagSNPs within the genomic region containing this miRNA in 695 adults with ADHD (266 and 396 subjects with and without comorbid SUD, respectively), 403 subjects with SUD without life-time diagnosis of ADHD and 485 sex-matched controls from Spain. Single and multiple marker analyses revealed association between two SNPs located at the 3′ region of miR-96 (rs2402959 and rs6965643) and ADHD without SUD. Our results provide preliminary evidence for the contribution of two sequence variants at the miR-183–96–182 cluster to ADHD without comorbid SUD, and emphasize the need to take comorbidities into account in genetic studies to minimize the effect of heterogeneity and to clarify these complex phenotypes.     

Small RNAs as Potential Platelet Therapeutics

MicroRNAs (miRNAs) are 21-23 nucleotide RNAs that regulate more than 60% of mammalian protein coding genes. miRNAs play critical roles in hematopoiesis and megakaryocyte function and development. Platelets, in addition to possessing functional miRNA processing machinery, have miRNA levels that have been correlated with platelet reactivity, and these miRNAs have been shown to target mRNAs that encode proteins that alter platelet function. There are potential uses of platelet miRNA as biomarkers and therapeutic agents. Due to the ability of platelets to release miRNA-containing microparticles at sites of activation, including angiogenic regions, tumors, and atherosclerotic plaques, there is the possibility of engineering platelets to deliver miRNA-based therapies to these sites. Cellpreferential expression of miRNAs could be exploited to restrict transgene expression in hematopoietic stem cell gene therapy to the desired lineage, including megakaryocytes and platelets. Finally, manipulation of gene expression in stored platelets may allow more effective platelet storage. Although much work remains to be done, there is great potential in miRNA-based platelet therapies.     

Invariant NKT cell development and function in microRNA-223 knockout mice

Invariant natural killer T (iNKT) cells, potent regulators of diverse immune responses, have been implicated in a number of diseases. The detailed mechanisms that drive iNKT cell development and maturation are still not completely understood. MicroRNAs (miRNAs) are small noncoding RNAs that regulate vast networks of genes that share miRNA target sequences. Our previous studies indicate that Dicer-dependent miRNAs play important roles in iNKT cell development, maturation, and function, but the roles of specific single miRNAs in this context are still lacking. Accumulated studies indicated that the miRNA miR-223 is a myeloid-specific miRNA. Here we report that miR-223 is highly expressed in thymic immature and activated splenic iNKT cells. To identify the role of miR-223 in iNKT cell development and function, miRNA-223-deficient mice were used. We have found that miR-223 deletion does not significantly interrupt iNKT cell development in the thymus, and miR-223-deficient mice have a normal frequency and number of iNKT cells in the thymus and peripheral immune organs. Furthermore, cytokine production of iNKT cells activated in vivo and in vitro shows no significant differences between miR-223 deficient mice and wild-type control. Thus, our data suggest that miR-223 may not be required for iNKT cell development and function.     

MicroRNA expression profiling of Chinese follicular lymphoma by microarray: A preliminary study

BackgroundMicroRNAs (miRNAs) have been widely regarded as crucial regulators in various biological processes involved in carcinogenesis. However, the comprehensive miRNA profiles of Chinese follicular lymphoma (FL) remains completely unknown.MethodsThe Exiqon miRCURY LNA™ microRNA Array (v.18.0) was used to detect the miRNA expression profiles of three Chinese FL samples, and compared to three reactive lymphatic nodes (RLN). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to confirm the selected miRNAs in different series. Three databases (miRAnda, miRBase and TargetScan) were used to predict the putative target genes. Bioinformatic analysis (gene ontology analysis and pathway analysis) was performed for further evaluation.ResultsThe microarray assay demonstrated that 1643 miRNAs were expressed; in which 103 miRNAs were upregulated and 68 miRNAs were downregulated, according to P-value (< 0.05) and fold change (FC > 2-fold). Furthermore, qRT-PCR was used to confirm that miR-17-5p, miR-20a-5p and miR-19a-3p were upregulated, and miR-3615 was downregulated (P < 0.05). Bioinformatic analysis (gene ontology analysis and pathway analysis) was used for further evaluation. Pathway analysis indicated that 25 pathways corresponded to differentially expressed miRNAs (P-value cut-off is 0.05). Furthermore, miR-17-5p, miR-20a-5p and miR-19a-3p were validated by qRT-PCR in an independent series including five FL3a and five RLN cases. Data analysis revealed that the changing trend of miR-19a-3p and miR-17-5p expression in the independent series was basically identical with that of the microarray data.ConclusionsOur results are the first to reveal the miRNA expression profiling of Chinese FL and three upregulated miRNAs. Furthermore, the expression of miR-19a-3p and miR-17-5p were found to be significantly upregulated in FL3a. Further study needs to be urgently performed to reveal its potential role in the pathogenesis of FL in the near future.