Skip to main content
. 2015 Oct 29;126(26):2871–2881. doi: 10.1182/blood-2015-02-631135

Figure 3.

Figure 3

miR-148a targets 3′UTR of TULA-2 mRNA and downregulates protein expression. (A) Schematic representation of miR-148a and 3′UTR of TULA-2 mRNA interaction. TULA2_148MUT construct was made by mutating 5 of the 7 nucleotides in the miR-148a binding seed region on TULA-2 3′UTR. Mouse TULA-2 mRNA (Ubash3b) varies by 2 nucleotides outside of the seed region from the human ortholog. (B) Luciferase reporter plasmids containing wild-type 3′UTR of TULA-2 or 148MUT was cotransfected into HCT116-Dicer-KO cells with β-galactosidase expression vector and miR-148a-3p mimic or scrambled miRNA control for 24 hours. Bar graph was plotted as normalized. (n = 3, *P < .05). (C) The 60 nM miR-148a-3p or control miRNAs were transfected into HCT-116-Dicer-KO cells or HEL cells by lipofectamine 2000 for 48 hours. TULA-2 protein level was blotted by western blotting. (D) HCT-116-Dicer-KO cells were transfected with 60 nM miR-148a-3p mimic, scrambled miRNA control, anti-miR-148a-3p LNA, or scrambled LNA control by lipofectamine 2000. RNA was isolated 48 hours after transfection, and TULA-2 mRNA expression was determined by quantitative RT-PCR (For miRNA overexpression, n = 3, *P < .05. for LNA transfection, n = 4, *P < .05). (E) HEL cells were transfected with 100 nM anti–miR-148a or scrambled anti-miR for 48 hours. TULA-2, total Syk, GPVI, and FcγRIIA protein level was detected by immunoblotting.