Figure 7.
Inhibition of TN/TX rescues loss of both miRNA and tumor suppressor activity in dicer haploinsufficiency.
KrasG12D; p53−/− mouse sarcoma cells encoding either dicer+/+ (KP) or dicer+/− (KP-D) were used to generate sub-cell lines stably expressing shRNA against luciferase (shluc), translin (shTN) or trax (shTX).
(A) Immunoblotting performed with extracts from KP and (B) KP-D cells.
(C) Pre-miRNA processing assays performed with 20 μg of extract from KP and (D) KP-D cells.
(E) Histogram summary of miRNA profiling studies. Data are presented as ratios of miRNA expression for shTN relative to shluc for KP (blue bars) and KP-D cells (red bars). These values represent the degree of miRNA suppression by TN/TX. The dashed line indicates equivalent miRNA levels for shTN and shluc. See also Table S5.
(F) Scatter plot representation of miRNA responsiveness to Dicer and TN/TX. Data are fold change for miRNA downregulated in KP-D versus KP cells (horizontal axis) and shTN versus shluc for KP-D cells (vertical axis). Closed circles represent miRNA with two-fold or greater decrease in KP-D versus KP. Open circles represent miRNA with less than two-fold decrease in KP-D versus KP. The correlation coefficient of the regression line for all data points is 0.89 (p < 0.0001).
(G) For proliferation studies, cells were plated, incubated overnight and baseline counts taken the next day (0 hours). Cell populations at 72 hours were normalized to those at 0 hours. * indicates higher cell counts for shluc versus shTN and shTX in KP-D cells (p < 0.003; n = 3; means ± SD).
(E) Cells were plated at clonal density (500 cells per 6-well plate) and incubated for 8 days. * indicates greater colony formation for shluc versus shTN and shTX in KP-D cells (p < 0.025; n = 3; means ± SD).
(F) Representative images of colony density at 2.5× magnification.