Figure 6: KSHV miRNAs directly repress the levels of the STAT3-regulated protein, BIRC5.
From: Viral microRNAs Target a Gene Network, Inhibit STAT Activation, and Suppress Interferon Responses
(A) HUVECs were transfected with individual KSHV miRNA mimics along with control miRNAs and the levels of BIRC5 were measured 48 hpt by qRT-PCR. (B) 3′UTR luciferase reporter assays were performed using pDEST-BIRC5-3′UTR (BIRC5-WT-3′UTR). 293 cells were reverse-transfected with wild type (WT) pDEST-BIRC5-3′UTR and individual KSHV miRNA mimics. Relative luciferase expression was measured at 24 hpt and 48 hpt as described in the Methods. (C) Binding site for KSHV miR-K12-5 as predicted by the target site prediction software, miRanda is shown (top). This site was mutated using site-directed mutagenesis (bottom) to generate mutant, pDEST-BIRC5-mut-3′UTR. 3′UTR reporter assays were repeated in 293 cells using pDEST-BIRC5—mut-3′UTR with miR-K12-5 and luciferase levels were analyzed at 24 hpt and 48 hpt. D. HUVECs were transfected with siRNAs targeting STAT3, BIRC5 or control siRNAs for 48 hours, following which they were infected with KSHV for 3 days. Total RNA was extracted from the cells and levels of LANA and GAPDH were measured by qRT-PCR.
