Figure 3
miR-155 and -181a target SAMHD1. (A) Quantification of miRNA levels. Total cellular microRNA was extracted from microglia and astrocytes and amplified for miRNA expression by qRT-PCR using specific primer for miR-155 and miR-181a. miRNA levels were normalized to U6 snRNA control and the cycle threshold (Ct) values were calculated. (B) Astrocytes (2 × 105 cells/ml) were transiently transfected with mimic or inhibitor for miR-155, miR-181a, and miR-146a, at a final concentration of 50 nmol. After 72 h, total cellular RNA was extracted and amplified by specific primer for SAMHD1 gene expression by RT-PCR. All data was normalized to GAPDH. Data shown represents fold change in mRNA levels. (C) Astrocytes (2 × 105 cells/ml) were transiently transfected with inhibitor for miR-155, miR-181a, or miR-146a individually or a combined transfection with both miR-155 and miR-181a inhibitors. Following transfection, SAMHD1 mRNA levels were quantified by RT-PCR. (D) Lysates were prepared from miR-181a inhibitor transfected cells and SAMHD1 protein expression was detected in corresponding cell lysates by immunoblotting with SAMHD1-specific antibodies. Equal amounts of total protein (30 μg) of the lysates from both cells were loaded. GAPDH was used as a loading control. (E) Astrocytes (2 × 105 cells/ml) were transiently transfected miR-181a inhibitor/mimic at a final concentration of 50 nmol. After 12 h, cells were infected with the HIV-luciferase pseudovirus (20 ng). At day 3, cells were harvested, washed with PBS, and lysed. Luciferase concentration in whole cell lysates was determined. Results shown are representative of mean ± SEM of three independent experiments (except for (D)). P value ≤0.05 were considered to be significant. SAMHD1, sterile alpha motif and histidine/aspartic acid domain-containing protein 1; VSV-G, vesicular stomatitis virus; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.