Figure 4

Investigation of the tumor suppressor function of miR-107 in PCa cells. (a) Cell proliferation analysis by miR-107 overexpression. Proliferation was significantly suppressed in MIA PaCa-2 cells transfected with the miR-107 mimic compared with cells transfected with the negative control mimic. The FACS analysis demonstrated that transfecting MIA PaCa-2 cells with the miR-107 mimic resulted in an accumulation of cells in the G0–G1 phase compared with transfection with the control miRNA mimic. In MIA PaCa-2 cells, p21 mRNA and protein levels were increased at 72 h after transfecting the cells with the miR-107 mimic. These findings indicated that miR-107 overexpression in PCa cells induced the production of p21, which results mainly in G0–G1 arrest. (b) miR-107 overexpression inhibits colony formation. Colony-formation assays were performed using MIA PaCa-2 cells. PCa cells were transiently transfected with the miR-107 or control mimic for two weeks. The number of colonies in MIA PaCa-2 cells treated with the miR-107 mimic was significantly lower than that in MIA PaCa-2 cells treated with the control mimic. (c) miR-107 overexpression induces cell apoptosis. The apoptotic cell analysis showed that miR-107 overexpression increased early apoptosis (annexin V-positive/PI-negative) and late apoptosis (annexin V/PI-double positive) 72 h after miR-107 mimic transfection compared with control mimic transfection in MIA PaCa-2 cells. (d) Notch2 as a novel target oncogene of miR-107 in PCa cells. An in silico search (http://www.targetscan.org/) identified Notch2 as a novel target oncogene of miR-107 in PCa. The seed regions of the miR-107 and complementary Notch2 3′UTR sequences are presented in this figure. miR-107 overexpression inhibited Notch2 protein production. Error bars indicate standard error of the mean (s.e.m.); n = 4 technical replicates of a representative experiment (out of four experiments).