GAS5 suppresses c-Myc translation through direct binding with its mRNA.
(A) The protein level of c-Myc was assessed by western blot after the cells were treated with GAS5 or control siRNA followed by the treatment of 100 µg/ml CHX for 1 and 2 hours. (B) c-Myc mRNA level associated with the polysome and non-polysome fractions of GAS5 and control siRNA transfected HEK-293T cells were evaluated by Q-PCR. Data was normalized with total mRNA (non-polysome + polysome). Bars represent mean ±SD from 3 replicates. (C) RNA from HEK-293T cells was pulled down by biotin-labeled GAS5 antisense DNA oligo with GAS5 sense DNA oligo as a control. The binding of c-Myc mRNA to GAS5 was evaluated by RT-PCR, in which GAPDH was used as a control.