Figure 2.
Potential regulatory function of miR-146b-5p on the expression of IL-12Rβ1. (A) The inhibitory effect of a given miRNA on a target sequence (3′UTR) expression was measured in cultured HEK293 cells. These cells were transfected with either the IL-23R 3′UTR or the 3' UTRI L-12Rβ1 together with miR mimics and miR control as indicated. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic and represent the mean of 5 and 7 experiments, respectively, carried out in triplicate. ∗P < .05. (B) Sequence alignment of miR-146b-5p and miR-146a-5p. The seed region on miRNAs and the potential target sequence on mRNA (site type 7mer-A1, Target scan release 7.2) are indicated in green. A further base pairing of miR-146b-5p with the 3′UTR IL-12Rβ1 is indicated in red. The position coordinates are indicated for the IL-12Rβ1 transcript isoform 3′ UTR: ENSG0000096996.11: ENST00000322153.7. (C) The insert sequence of 3′ UTR clone NM_153701 (Origene, cod SC208722) (IL-12Rβ1 3' UTR-WT) and the same insert deleted of the sequence GTTCTCA complementary to the seed sequence (nt328-nt334, Origene) (IL-12Rβ1 3′ UTR-MUT). Blue, stop codon; red, cloning site; highlighted in green, the seed sequence. (D) HEK 293 cells were transfected with either the IL-12Rβ1 3′UTR-WT or with IL-12Rβ1 3′UTR-MUT with miR mimics and miR control. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic (reference line at 100%) and represent the mean of 4 experiments carried out in triplicate. P value is statistically significant (P < .05, t test). (E) Western blotting analysis of IL-12Rβ1 and GAPDH or β-Actin expression in purified CLL cells from 7 different cases transfected with miR-146b-5p inhibitor or miR-CTR inhibitor and cultured for 48 hours. (F) Fold change values of the IL-12Rβ1 signal normalized to that of GAPDH or β-Actin of cells transfected with miR-146b-5p inhibitor/IL-12Rβ1 normalized signal of cells transfected with miR-CTR inhibitor. Percentage (%) changes in the relative abundance of IL-12Rβ1 protein are also indicated for each CLL sample and calculated as (fold change − 1) × 100. A positive percentage indicates increased abundance relative to the control. (G) Summary of the results of the experiments in (F). Protein bands from immunoblotting were analyzed using ImageJ Analysis Software or by Alliance LD, UVITEC. Data are presented as IL-12Rβ1/GAPDH or IL-12Rβ1/β-Actin (mean ± SD). The P value of the difference between CLL cells treated with miR-146b-5p vs miR-CTR inhibitors is indicated (Wilcoxon test). ∗P < .05.

Potential regulatory function of miR-146b-5p on the expression of IL-12Rβ1. (A) The inhibitory effect of a given miRNA on a target sequence (3′UTR) expression was measured in cultured HEK293 cells. These cells were transfected with either the IL-23R 3′UTR or the 3' UTRI L-12Rβ1 together with miR mimics and miR control as indicated. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic and represent the mean of 5 and 7 experiments, respectively, carried out in triplicate. ∗P < .05. (B) Sequence alignment of miR-146b-5p and miR-146a-5p. The seed region on miRNAs and the potential target sequence on mRNA (site type 7mer-A1, Target scan release 7.2) are indicated in green. A further base pairing of miR-146b-5p with the 3′UTR IL-12Rβ1 is indicated in red. The position coordinates are indicated for the IL-12Rβ1 transcript isoform 3′ UTR: ENSG0000096996.11: ENST00000322153.7. (C) The insert sequence of 3′ UTR clone NM_153701 (Origene, cod SC208722) (IL-12Rβ1 3' UTR-WT) and the same insert deleted of the sequence GTTCTCA complementary to the seed sequence (nt328-nt334, Origene) (IL-12Rβ1 3′ UTR-MUT). Blue, stop codon; red, cloning site; highlighted in green, the seed sequence. (D) HEK 293 cells were transfected with either the IL-12Rβ1 3′UTR-WT or with IL-12Rβ1 3′UTR-MUT with miR mimics and miR control. Both firefly luciferase and Renilla luciferase activities were measured after a 48-hour culture. Data shown are relative to the reporter vector transfected with the miR CTR mimic (reference line at 100%) and represent the mean of 4 experiments carried out in triplicate. P value is statistically significant (P < .05, t test). (E) Western blotting analysis of IL-12Rβ1 and GAPDH or β-Actin expression in purified CLL cells from 7 different cases transfected with miR-146b-5p inhibitor or miR-CTR inhibitor and cultured for 48 hours. (F) Fold change values of the IL-12Rβ1 signal normalized to that of GAPDH or β-Actin of cells transfected with miR-146b-5p inhibitor/IL-12Rβ1 normalized signal of cells transfected with miR-CTR inhibitor. Percentage (%) changes in the relative abundance of IL-12Rβ1 protein are also indicated for each CLL sample and calculated as (fold change − 1) × 100. A positive percentage indicates increased abundance relative to the control. (G) Summary of the results of the experiments in (F). Protein bands from immunoblotting were analyzed using ImageJ Analysis Software or by Alliance LD, UVITEC. Data are presented as IL-12Rβ1/GAPDH or IL-12Rβ1/β-Actin (mean ± SD). The P value of the difference between CLL cells treated with miR-146b-5p vs miR-CTR inhibitors is indicated (Wilcoxon test). ∗P < .05.

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