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Anthony T Nguyen, Zeljka Smit-McBride, Matthew Bordbari, Amar P Patel, David Gregory Telander, Lawrence S Morse; Circulatory MicroRNAs in Serum as Potential Biomarkers for AMD. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1328.
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© ARVO (1962-2015); The Authors (2016-present)
The circulatory microRNAs in serum have recently been identified as novel biomarkers in a number of important chronic diseases. The aim of our study was to identify serum microRNA biomarkers for dry AMD and wet AMD compared to controls.
Serum samples from 40 patients of 4 different groups were collected. Group 1 were 10 age-matched normals with no ocular or systemic disease, Group 2 were 10 patients with category 3 high risk dry AMD, Group 3 were 10 new unilateral exudative AMD patients treated with Lucentis or Avastin, and Group 4 were 10 patients resistant to anti-VEGF therapy. RNA was isolated using modified Qiagen microRNeasy procedure and quantitated on BioAnalyzer’s small RNA chips. Microarray miRNA analysis of serum samples was performed on 4 samples from each group and ran on Affymetrix 2.0 miRNA arrays. Statistical analysis was done by ANOVA. Confirmatory quantitative real-time PCR (qPCR) using Exiqon's LNA™ Universal RT microRNA PCR, was done on the select set of biomarker candidates for the full sample set of each group. RNA spike-ins and melting curve analysis served as quality control, as well as serial dilutions to detect possible bias. Three microRNAs with stable expression in serum served as reference genes for data normalization. ANOVA was used for statistical analysis.
Many statistically significant miRNA differences were seen between the 4 groups involving both increased and decreased levels. Total number of miRNAs with statistically significant differences in expression (p<0.01) are listed. The miRNAs showing largest dysregulations are listed in parentheses - up-regulated are listed first for each group comparison, followed by the down-regulated. Dry AMD vs. control had 10 (miR-661, miR-3121), wet AMD vs. control had 9 (miR-4258, miR-889), and resistant AMD vs. control had 9 (miR-438, miR-3261). Subsequent qPCR on the full sample sets showed a 6.8 fold increase in one of the members of the let-7 miRNA family, when Group 3 patients were compared to Group 4 patients (p<0.01). This family of miRNAs has been shown to be involved in the regulation of angiogenesis.
Our study indicates several potential biomarkers for dry and wet AMD, as well as potential biomarkers of resistance to anti-VEGF treatment. MiRNA levels that were significantly altered between the groups tested may offer diagnostic and therapeutic benefits.
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