miR-16 directly targets BCL2 and decrease BCL2 expression. (A) analyzing the homology between miR-16 and BCL2 mRNA sequences. (B) Luciferase assay revealed reduced relative luciferase activities in 293T cells stably over-expressing miR-16 following transfection of BCL2 3’-UTR using pMIR and pMIR-REPORT vectors (P<0.01). FL, firefly luminescence; RL, Renilla luminescence. (C) Quantitative RT-PCR analysis showed that miR-16 inhibited the expression of BCL2. *P < 0.05; **P < 0.01. (D) Western blot analysis showed that miR-16 led to obvious decrease of BCL2 expression.
miR-16 increase apoptosis. (A) Lysates from PTC cells treated with miR-16 for 24 h were analyzed. Levels of cleaved caspase-8, caspase-9, caspase-3, and PARP were detected and compared with the total levels of each protein (labeled with C vs. T). Detection of Actin was performed as a loading control. (B) PTC cell cycle was analyzed by ﬂow cytometer (PI staining) 48 h post transfection. In the group of miR-16, the proportion of cells at G0/G1 stage was much higher than negative groups (P<0.05). (C) Apoptosis was detected in the negative and miR-16 group of PTC cells by flow cytometry. (D) Quantification of the flow cytometry data according to apoptosis stage is shown with the error bars representing standard deviation values. P < 0.01, based on the Student’s t-test.
miR-16 decreases BCL2 expression through ERK1/2 pathway. (A) Detection of the protein amount of BCL2, phosphorylated ERK1/2, and total of ERK1/2 in day-1, day-3 and day-5 PTC cells with miR-16 treatment by western blotting assay. The cells with no miR-16 and no PD98059 treatment. The expression level of BCL2 in control group have no obvious change with the time increased. (B) The cells treat with miR-16 but no PD98059, miR-16 decrease BCL2 expression for 3 days and 5 days. Concurrently, a lower expression level of phosphorylated ERK 1/2 is detected. (C) The cells treat with miR-16 and PD98059, the expressions of BCL2 and phosphorylated ERK1/2 are significantly inhibited by PD98059. In contrast, there is no significant change in the total amounts of ERK. P < 0.01, based on the Student’s t-test.