Figure 5From: microRNA-29 negatively regulates EMT regulator N-myc interactor in breast cancerUpregulation of miR-29 as a consequence of compromised NMI expression. (A) ChIP-data reported by the ENCODE project confirms the existence of a TCF site in the upstream regulatory region of miR-29 a and b1 on chromosome 7. (B) T47D cells grown in growth factor free medium were treated with Wnt3a ligand (100 ng/ml) or insulin (10 μg/ml) for 24 hr. Levels of miR-29 a/b using RT-PCR. The levels were compared to untreated cells (control), set arbitrarily at 100%. (C) T47D-shNMI cells (stably silenced for NMI) were evaluated for β-catenin driven transcription activity (using TOP-FLASH reporter) at basal levels or with Wnt3a (100 ng/ml) treatment. This activity was compared to the T47D-scrambled control cells. Error bars represent ± SEM and * indicates P ≤ 0.05. (D) Total RNA from T47D-shNMI cells was analyzed for levels of miR-29a and b and compared with T47D-scrambled control cells. Introduction of constitutively active GSK3β (CA-GSK3β) in T47D-shNMI resulted in significant reduction of miR-29 expression. Error bars represent ± SEM and * indicates P ≤ 0.05. (E) Total protein from T47D-shNMI and corresponding scrambled control cells was analyzed for the levels of total GSK3β and phospho-Ser-9-GSK3β using western blot analysis. Level of NMI confirmed the stable silencing of NMI in these cells and GAPDH was used as a loading control. (F) Proposed model of miR-29 signaling: In absence of NMI and/or due to micro-environmental signals such as Wnt or insulin, GSK3β is inactivated (upon phosphorylation at Ser 9). That leads to increased miR-29 levels possibly by elimination of check on TCF driven transcription. miR-29 in turn targets NMI, which is one of the factors responsible to guard the active status of GSK3β; thus driving a feed forward loop for miR-29 activation and sustained loss of NMI. This signaling mechanism may be one of the promoters of invasive progression of breast cancer.Back to article page