MicroRNA profiling of Oropharyngeal tissue from chronically SIV infected rhesus macaques

The study describes miRNA expression in Oropharyngeal tissue of chronically SIV-infected rhesus macaques. To identify the underlying molecular mechanisms we simultaneously profiled miRNA and mRNA expression in oropharyngeal tissues of chronically simian immunodeficiency virus (SIV)-infected rhesus macaques (RMs). Relative to controls, we identified 48 (38-upregulated and 10 downregulated) differentially expressed (DE) miRNAs relative to uninfected controls (n=5). Interestingly, in terms of magnitude, miR-19a, miR-301, miR-142-3p, miR-32 and miR-142-5p were among a select list of miRNAs that showed the highest upregulation in OPM. An important finding is the significant upregulation in OPM of miR-21, a microRNA known to regulate periodontitis, T-cell activation and oral carcinoma. Interestingly, RNA-seq for gene expression profiling also confirmed miR-21 upregulation in OPM of VEH-untreated/SIV rhesus macaques. Using TargetScan 7.2, we identified TSC22D3 (TSC22-domain family member 3), an anti-inflammatory protein induced by glucocorticoids and IL10 that was significantly downregulated in OPM of VEH-untreated/SIV macaques to be a predicted target of miR-29b. TSC22D3 localized to minor salivary gland acini and secretory ducts and showed reduced expression in OPM of VEH-untreated chronically SIV macaques. Using luciferase reporter and overexpression assays, we confirmed TSC22D3 to be a direct target of miR-29b. Interestingly, expression of miR-150 was significantly downregulated, a miRNA we previously demonstrated to be downregulated during T cell activation in the intestine. These findings strongly support a role for differential miRNA expression associated with HIV/SIV induced oropharyngeal mucosal dysfunction. Twelve age and weight matched male Indian rhesus macaques were randomly divided into 2 groups. Group 1 (n=5) remained uninfected. Group 2 (VEH-untreated/SIV, n=7) animals were infected intravenously with 100TCID50 of SIVmac251. Oropharyngeal tissue was collected at necropsy, ~180 days post SIV infection. ~100 ng of total RNA was first reverse transcribed and preamplified according to the manufacturer’s recommendation. MicroRNA expression profiling was performed using TaqMan ®OpenArray® Human microRNA panels. Data analysis was performed using ExpressionSuite® software, which utilizes the comparative Cτ (ΔΔCτ) method to rapidly and accurately quantify relative miRNA expression across many genes and samples. miRNA expression data were normalized using a combination of snU6, RNU44 and RNU48. Comparisons were made between uninfected and VEH-untreated SIV-infected rhesus macaques.