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. 2016 Feb 26;11(2):e0149954. doi: 10.1371/journal.pone.0149954

Fig 1. IRS-2 is a potential target of miR-126 in INS-1 β cells.

Fig 1

(A)Bioinformatics analysis indicated that there was a 6 consecutive nucleotide seed sequences as the potential miR-126 binding site in the 3’ -UTR region of IRS-2 mRNA, as indicated with short vertical lines. (B) A 199bp fragment of IRS-2 3’-UTR region was cloned into psicheck-2 reporter plasmid. Then the luciferase reporter assay was performed by co-transfection of the luciferase reporter vector and the miR-126 mimic (126M), with the nonspecific sequence (NC) as the negative control. A complementary miR-126 inhibitor (126I) was designed to specifically block the miR-126, both endogenously and exogenously. Then the luciferase reporter assay was re-performed with the mutant reporter vector, co-transfected with the nonspecific sequence (NC), miR-126 mimic (126M), miR-126 inhibitor (126I) respectively. Luciferase activity was tested 24 h after transfection. (C) The IRS-2 -psicheck-2 reporter plasmid was mutated at the center of the miR-126 seed sequences. (D, E) The expression of the IRS-2 protein was detected by western blot. The relative levels were normalized with the GAPDH and represented as a column chart.