Figure 3.

miR-183 targets the 3ʹUTR of MICA/B mRNA. (a) Schematic of luciferase reporter constructs carrying wild-type (WT) MICA or MICB 3ʹUTR as well as mutated (MUT) MICA or MICB 3ʹUTR. The miR seed sequences are depicted as capital letters and the mutated nucleotides are in red. CMV, cytomegalovirus promoter; LUC, luciferase. (b) 293T cells were transfected with luciferase constructs containing MICA-WT, MICA-MUT, MICB-WT or MICB-MUT (500ng), renilla luciferase (5ng) together with miR-183 (25nM). After 24 h, the cells were lysed and quantified for firefly luciferase activity. (c) Lentiviral constructs containing scramble control or anti-sense miR-183 were transfected into H1355 lung tumor cells. qPCR analysis was conducted to confirm the downregulation of miR-183 expression by antisense miR-183 (d, e) Flow cytometric analysis using a monoclonal antibody that detects a common sequence in MICA and MICB indicated that antisense miR-183 transfected H1355 tumor cells upregulated MICA/B expression. D is a representative staining of MICA/B. (f, g, h) H1299 lung tumor cells also transfected with antisense miR-183 showed upregulation of both MICA mRNA by qPCR and protein by flow cytometry analysis. G, H is a representative staining of MICA/B. Each of the experiments is representative of at least 3 experiments performed.