MicroRNA-16 Is Down-Regulated in Mutated FLT3 Expressing Murine Myeloid FDC-P1 Cells and Interacts with Pim-1
Figure 6
Mir-16 mimic down-regulates Pim-1 and decelerates FD-FLT3/ITD cell growth.
(A) Using QPCR, Pim-1 mRNA level was quantified from FD-FLT3/ITD cells upon miR-16 mimic transfection. The graph is presented to show relevant gene expression fold differences in FD-FLT3/ITD cells after miR-16 mimic transfection compared to scrambled miR-16 transfection (fold = 1) as a control. The results show the mean and standard deviation for triplicate QPCR results from two independent experiments (p<0.01). The inset shows the results from immunoblotting of Pim-1 protein upon transfection of miR-16 mimic. Total protein extracts were obtained from FD-FLT3/ITD cells and then subjected to size fractionation in 10% poly-acrylamide gels. Immunoblotting was performed and probed with anti-Pim-1 antibody. Pim-1 protein expression has been reduced in FD-FLT3/ITD cells upon miR-16 mimic transfection (lane 2) when compared to control Pim-1 protein expression (lane 1). The membrane was stripped and re-probed with anti- β-actin antibody to demonstrate equal loading of total protein extract. (B) Overexpression of miR-16 mimic decelerates growth of FD-FLT3/ITD cells. A quantity of 1×106 FD-FV and FD-FLT3/ITD cells were cultured after transfection with synthetic miR-16 mimics for 4 days. Viable cells were counted daily on the basis of trypan blue exclusion. Results shown are the means from triplicate assays. Error bars indicate SD.