miR-Sens—a retroviral dual-luciferase reporter to detect microRNA activity in primary cells

Validation of miRNA activity in LATS2 and TXNIP 3′ UTRs in the absence of PAS. (A) Relative luciferase activities in BJ control or miR-371-2-3 cluster expressing cells for miR-372-3p and -373-3p sensors. (B) LATS2 (ENST00000382592) and TXNIP (ENST00000369317) 3′ UTRs. (Black rectangles) miR-372 and miR-373 binding sites; (gray rectangle) miR-31 site; (black cross) miRNA-binding site mutation of miR-372 and miR-373 (Voorhoeve et al. 2006); (373/3WT and 372/3MUT) wild-type and double miR-372 and miR-373 mutant binding sites in LATS2 3′ UTR; (31MUT) miR-31 mutant binding site in LATS2 3′ UTR; (AATAAA) alternative PAS for LATS2 3′ UTR (EST ACC#BQ009415). We have confirmed that only the most 3′ LATS2 PAS was used in BJ cells by 3′ RACE PCR (data not shown). (C) Relative luciferase activities in BJ control or miR-371-2-3 cluster expressing cells for LATS2 short wild-type and double-mutant 3′ UTRs. (D) Relative luciferase activities in BJ control or miR-371-2-3 cluster expressing cells for LATS2^wt and TXNIP 3′ UTRs in the absence of the most 3′ A(A/T)AAA sequence (Table 2). Comparable decrease of the relative luciferase activity was obtained when including the most 3′ A(A/T)AAA sequence of LATS2 and TXNIP 3′ UTRs in BJ cells and (E) AGS cell line. In these experiments, the relative luciferase activity of the miR-Sens vectors in control cells is normalized to 100% (double-normalization).