Figure 3
Figure 3. miRNA-494 and miRNA-320a affect survivin expression and apoptosis. (A) Bioinformatic target prediction analysis identifies survivin as a target of miR-494 and miRNA-320a; the survivin 3′ UTR complementary to the miRNA sequence is shown (using Targetscan). (B) Both miRNA-494 and miRNA-320a can regulate survivin expression. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and survivin expression was analyzed by Western blot analysis using a rabbit anti-survivin antibody (Cell Signaling Technology). The miRNA with no human targets were used as control. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (C) miRNA-494 and miRNA-320a induce apoptosis. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and apoptosis was assessed by FACS analysis of annexin V–FITC/PI staining of control-treated (miRNA with no human targets, see “Methods”), and mimics for miRNA-494 and miRNA-320a. Data are shown from 1 of 3 representative experiments and indicate early apoptosis (annexin V single-positive cells). Percentages of annexin V–positive cells are indicated in the figure (SEs are under 2%). Percentages refer to the cells within the top left and bottom left quadrants, and top right and bottom right, respectively. (D) TEL-AML1 regulation of survivin is Dicer1 dependent. siRNA silencing of Dicer1 restores survivin expression in TEL-AML1 depleted cells indicating the miRNA dependence of this effect. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (E) Luciferase vectors containing portions of the survivin (BIRC5) 3-UTR was used to demonstrate that survivin is a direct target of miRNA-494 and miRNA-320a. Both miRNAs were able to block luciferase expression significantly when they were cotransfected into REH cell together with the BIRC5 3-UTR luciferase-expressing vector (WT BIRC5). A mutated sequence and the empty vector were used as controls (MUT BIRC5 and pMir Reporter, respectively), and both had equivalent levels of luciferase activity. The Y-axis displays relative luminescence units, which are normalized per unit of beta-galactosidase activity. SEs from 3 separate experiments are shown. The wild-type target sequence had at minimum average 6-fold lower luminescence compared with the mutant targets, and miRNA targeted to the mutant sequence did not significantly impact luciferase activity compared with the empty vector control. Sequences used to create the plasmids are shown in supplemental Table 1. The mismatch mutant vectors contain 2 mismatch bases from the miRNA seed sequence.

miRNA-494 and miRNA-320a affect survivin expression and apoptosis. (A) Bioinformatic target prediction analysis identifies survivin as a target of miR-494 and miRNA-320a; the survivin 3′ UTR complementary to the miRNA sequence is shown (using Targetscan). (B) Both miRNA-494 and miRNA-320a can regulate survivin expression. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and survivin expression was analyzed by Western blot analysis using a rabbit anti-survivin antibody (Cell Signaling Technology). The miRNA with no human targets were used as control. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (C) miRNA-494 and miRNA-320a induce apoptosis. miRNA mimics for miRNA-494 and miRNA-320a were transfected into the REH cells and apoptosis was assessed by FACS analysis of annexin V–FITC/PI staining of control-treated (miRNA with no human targets, see “Methods”), and mimics for miRNA-494 and miRNA-320a. Data are shown from 1 of 3 representative experiments and indicate early apoptosis (annexin V single-positive cells). Percentages of annexin V–positive cells are indicated in the figure (SEs are under 2%). Percentages refer to the cells within the top left and bottom left quadrants, and top right and bottom right, respectively. (D) TEL-AML1 regulation of survivin is Dicer1 dependent. siRNA silencing of Dicer1 restores survivin expression in TEL-AML1 depleted cells indicating the miRNA dependence of this effect. For loading controls, an equal number of cells from each experimental sample were lysed, and the same volume of the same lysate was applied to a separate gel to probe for tubulin. (E) Luciferase vectors containing portions of the survivin (BIRC5) 3-UTR was used to demonstrate that survivin is a direct target of miRNA-494 and miRNA-320a. Both miRNAs were able to block luciferase expression significantly when they were cotransfected into REH cell together with the BIRC5 3-UTR luciferase-expressing vector (WT BIRC5). A mutated sequence and the empty vector were used as controls (MUT BIRC5 and pMir Reporter, respectively), and both had equivalent levels of luciferase activity. The Y-axis displays relative luminescence units, which are normalized per unit of beta-galactosidase activity. SEs from 3 separate experiments are shown. The wild-type target sequence had at minimum average 6-fold lower luminescence compared with the mutant targets, and miRNA targeted to the mutant sequence did not significantly impact luciferase activity compared with the empty vector control. Sequences used to create the plasmids are shown in supplemental Table 1. The mismatch mutant vectors contain 2 mismatch bases from the miRNA seed sequence.

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