MicroRNA - Volume 4, Issue 1, 2015

Glaucoma targets a variety of different tissues located in both anterior (e.g., trabecular meshwork) and posterior (e.g., optic nerve head) ocular segments. The transmission of damage between these structures cannot be simply ascribed to intraocular pressure increase. Recent experimental findings provide evidence for the involvement of molecular mediators including proteins and microRNAs. Aqueous humor protein composition is characteristically altered during glaucoma progression. Immunohistochemistry analyses indicate that proteins characterizing glaucomatous aqueous humor are released by damaged trabecular meshwork. This feature incudes (a) Nestin, involved in stem cell recruitment and glial cell activation; (b) A Kinase anchor protein, released as consequence of mitochondrial damage and Rho activation establishing cell shape and motility; (c) Actin related protein 2/3 complex, involved in actin polymerization and cell shape maintenance. As established both in vitro and in glaucomatous aqueous humor, trabecular meshwork cells damaged by oxidative stress release extracellular microRNAs inducing glial cell activation, an established pathogenic mechanism in neurodegenerative diseases. Released microRNAs include miR-21 (apoptosis), miR-450 (cell aging, maintenance of contractile tone), miR-107 (Nestin expression, apoptosis), miR-149 (endothelia and extracellular matrix homeostasis). Experimental evidences indicate that the uveoscleral pathway, via suprachoroidal space, can provide a potential route of access from the anterior region to the posterior segment of the eye and could represent the path followed by biologic mediators to reach the inner layer of the peripapillary retina and transmit damage signals from the anterior to posterior segment during glaucoma course.

MicroRNAs (miRNAs) are small stable endogenous RNAs, found in all complex organisms and considered by nature as inhibitors of mRNA translation. This class of small posttranscriptional regulatory RNAs originates from the random formation of hairpin precursors in “non-coding” DNA regions; their main function is the control of gene expression status. The premature forms of the fullydeveloped microRNAs are the pre-microRNAs; molecules composed of thousands of nucleotides and constitute 1-2% of the human genome; the human genome encodes at least 1,500 miRNAs. miRNAs do not encode for any proteins; they guide gene modulation and affect crucial biological processes such as cell proliferation, tissue differentiation, apoptosis, cancer progression and female physiology. miRNAs are detected in specific tumor types and seem to be effectively involved in invasion, metastasis and acquire a role in clinical prognosis. miRNA expression is the vital key in cancer cell dysregulation. Cancer cells present with abnormal growth and lack of their apoptosis fate. Normal and neoplastic tissues have dissimilar expression patterns. Endometrial cancer subtypes are outlined by an irregular miRNA expression, along with other human malignancies. miRNAs retain manifold roles in angiogenesis; have a bidirectional impact on oncogenes and tumor suppressor genes in the steps of endometrial cancer growth, being positive or negative regulators of metastasis. The scientific significance of this new class of noncoding RNAs is gradually comprehended. The current review summarizes our knowledge on the role of miRNA in endometrial cancer development and clinical behavior; it outlines the biological pathways strictly modulated by them.

New therapies based on the targeting of signal pathways (such as VHL/HIF-1) common to most renal cell carcinomas (RCC), have greatly improved the outlook for sufferers of this disease. Given the growing reputation of many microRNAs (miRNAs) as both tumor suppressors and oncogenes, the measurement and manipulation of these small nucleotides in RCC patients may provide yet another valuable advance in renal cancer diagnosis and treatment. The present review summarizes the current literature on the role of microRNAs in RCC development and progression emphasizing the interaction of specific miRNAs with both oncogenic and tumor suppressor pathways of particular importance in renal cancer.

MicroRNAs (miRs) have emerged as useful biomarkers for different disease states, including allergic inflammatory diseases such as asthma and eosinophilic esophagitis (EoE). Serum miRs are a possible non-invasive method for diagnosis of such diseases. We focused on microRNA-21 (miR-21) levels in serum, in order to assess the feasibility of using this gene as a non-invasive biomarker for these diseases in the clinic, as well as to better understand the expression pattern of miR-21 in allergic inflammation. We used quantitative PCR (QPCR) to assay miR-21 and other control miRs in esophageal biopsies from EoE patients and serum samples from EoE and asthma patients. Serum levels of miR-21 were significantly elevated in patients with asthma, whereas serum miR-21 levels were not associated with the presence of allergen-specific IgE (i.e. atopy). Esophageal biopsies showed a large elevation of miR-21 in EoE and an increase in miR-21 in EoE serum. Control U6 miR did not vary between asthma and control patients, however EoE serum had significantly decreased U6 microRNA compared to controls. The decreased U6 in EoE sera did not completely account for the relative increase in miR-21 in the sera of EoE patients. We report for the first time that miR-21 is elevated in the sera of both asthma and EoE patients. We find no relation between serum miR-21 levels and atopy. Our results thus suggest miR-21 is a novel biomarker for human allergic inflammatory diseases.

MiRNAs are gene (post-transcriptional) regulators that bind the 3’UTR of target genes. Single-nucleotide polymorphisms (SNPs) located within a miRNA binding site can impact miRNAdependent gene regulation by weakening or reinforcing the microRNA:mRNA bond. We present a general in silico approach enabling researchers to “predict” which of the several SNPs of 3’UTR of H2AFX gene can mainly affect its regulation. H2AFX gene encodes a member of the H2A histone family which is central in the detection of and response to DNA double-strand breaks. All the 17 common SNPs located within the 3’UTR of H2AFX gene were analyzed for putative miRNA-binding sites by using different databases (such as dbSNP and miRBase) and pre-existing algorithms (such as MicroSNiPer and RNAcofold) in order to calculate the minimum free energies of hybridization of the microRNA:mRNA duplex, for both the wild-type and mutant alleles. The difference in these energies was also calculated. Since in each tissue one target sequence can bind only one miRNA at a time, the sum of all the difference of energies can be considered a relevant parameter for predicting the importance of a SNP with respect to miRNA regulation. We used tertiles to classify the SNPs and provide a priority list based on their theoretically strongest impact on miRNA binding. By using the described approach, we provided the basis for a reasoned, user-friendly algorithm-driven selection of SNPs impacting miRNA biology. The proposed method is helpful for selecting SNPs having a more powerful (putative) biological function, minimizing workflow and costs for experimental and clinical investigations.

Mutations of the FOXC1 transcription factor are involved in a variety of autosomal dominant ocular anterior segment defects, ranging from Axenfeld-Rieger malformations to isolated glaucoma in some patients. In this study we have evaluated the possible role of the c.*734A>T FOXC1 variant as a modifier factor of the activity of two FOXC1 mutations previously identified in families primarily affected by dominant glaucoma (haplotypes p.G447_G448insDG-c.*734A>T and p.I126S-c.*734A>T). Previous bioinformatic analyses indicated that the c.*734A>T variant is located in a potential target sequence for hsa-miR-548l. Co-expression of this miRNA with a reporter cDNA construct in which the wild-type 3’UTR sequence of FOXC1 was fused to the 3’-end of the firefly luciferase coding region, led to approximately 20% decreased luciferase activity compared to the controls, confirming the presence of a target sequence for hsa-miR-548l. In contrast, this miRNA did not show any effect on the luciferase activity associated with the mutant 3’UTR FOXC1 sequence, showing that it resulted in a loss-of-function of the has-miR-548l target sequence. In addition, functional evaluation of the two glaucoma-associated haplotypes revealed increased protein levels and transactivation, compared to the corresponding individual coding mutations (approximately 1.2-fold on average). These data support the role of hsa-miR-548l as a regulator of FOXC1 translation and provide evidence for the c.*734A>T variant as a modifier factor for the activity of coding glaucoma-associated FOXC1 mutations.

Background: Atherosclerosis-related cardiovascular disease and osteoporosis (OP) occur concurrently and may share a common pathogenesis. Aberrant expression of miR-21 and vitamin D deficiency have been independently linked to the pathogenesis of atherosclerosis and OP. Objectives: To examine the relationship between miR-21 expression and vitamin D in aorta and bone in atherosclerotic disease. Methods: Aorta, internal mammary artery (IMA) and sternal bone samples were collected from patients undergoing coronary artery bypass graft (CABG) surgery. Bone density was measured by dual x-ray absorbtiometry (DXA). MiR-21 was quantified using a two-step reverse transcription-polymerase chain reaction. Results: Ten patients were included for analysis; 5 were vitamin D deficient (<25nmol/L). MiR-21 was expressed at a greater level in aorta compared with the IMA (p = 0.003), and sternal bone (p = 0.002). Expression of miR-21 between the IMA and bone was similar (p = 0.7). A positive correlation between the magnitude of difference (fold-difference) of miR-21 expression between aorta and IMA and CRP (correlation coefficient 0.9, p = 0.009) was found. Vitamin D deficient patients had greater expression of miR-21 in aorta compared with non-deficient patients (p = 0.03). Increasing CRP and vitamin D deficiency were independent predictors of miR-21 expression in aorta. The lower the difference in miR-21 expression between aorta and bone, the lower the bone density. Conclusion: In atherosclerosis, miR-21 is increased in the aorta and associated with vitamin D deficiency. Vitamin D deficiency may influence aberrant miR-21 expression in vasculature and bone contributing to the concurrent development of atherosclerosis and osteoporosis.

Clefting of the secondary palate is the most common birth defect in humans. Midline fusion of the bilateral palatal processes is thought to involve apoptosis, epithelial to mesenchymal transition, and cell migration of the medial edge epithelium (MEE), the specialized cells of the palate that mediate fusion of the palatal processes during fetal development. Data presented in this manuscript are the result of analyses designed to identify microRNAs that are expressed and regulated by TGFβ3 in developing palatal MEE. The expression of 7 microRNAs was downregulated and 1 upregulated in isolated MEE from wildtype murine fetuses on gestational day (GD) 13.5 to GD14.5 (prior to and during epithelial fusion of the palatal processes, respectively). Among this group were miRNAs linked to apoptosis (miR-378) and epithelial to mesenchymal transformation (miR-200b, miR-205, and miR-93). Tgfβ3-/- fetuses, which present with a complete and isolated cleft of the secondary palate, exhibited marked dysregulation of distinct miRNAs both in the palatal MEE and mesenchyme when compared to comparable wild-type tissue. These included, among others, miRNAs known to affect apoptosis (miR-206 and miR-186). Dysregulation of miRNAs in the mesenchyme underlying the palatal MEE of Tgfβ3-/- fetuses is also discussed in relation to epithelial-mesenchymal transformation of the MEE. These results are the first systematic analysis of the expression of microRNAs in isolated fetal palatal epithelium and mesenchyme. Moreover, analysis of the Tgfβ3 knockout mouse model has enabled identification of miRNAs with altered expression that may contribute to the cleft palate phenotype.