FIGURE 6.

Knocking down miR-185-3p increases c-Myc level, promotes c-Myc transcriptional activity, and thus facilitates serum-induced proliferation. Knocking down miR-185-3p increases the c-Myc level in HCT116 (A) and MCF7 (B) cells. HCT116 and MCF7 cells were double transfected with the indicated miRNA inhibitors, and cells were harvested 24 h after the second transfection and subjected to immunoblot with antibodies as indicated (top panels) and real-time PCR for the assessment of the miR-185-3p and E2F2 mRNA levels (bottom panels). c-Myc was induced at 6 h after changing medium of cultured cells. C, inhibition of miR-185-3p prolongs the serum induction of c-Myc in cells. HCT116 cells were double transfected with indicated miRNA inhibitors or negative control (CT). 6 h after the second transfection, the cells were stimulated by changing culture medium to 20% FBS fresh medium and harvested at the indicated time points. Cell lysates were subjected to Western blot (WB) analysis with the indicated antibodies. This assay was repeated three times. Bottom, quantification of the results from the assays as shown in the top as a representative graph here. The y axis represents the -fold increase of c-Myc protein levels, and the x axis denotes the time course after serum stimulation. A t test was performed with p < 0.05 for the 8 h time point (n = 3). D, miR-185-3p inhibitor, but not control inhibitor, facilitates serum-induced cell proliferation. Cells were treated the same way as in C and labeled with BrdU 2 h before harvesting and then subjected to anti-BrdU staining (red), as shown on the left. The percentage of BrdU-positive cells is shown on the right. Significance was determined using an unpaired Student's t test. *, p < 0.05. Data are presented as means ± S.E. (error bars), n = 3. E, a model for the c-Myc-miR-185-3p feedback loop.