Polyclonal anti-Sirt-1 and antiNCoR were purchased from Abcam PLC

on 32 14 Overexpression rate 52.46 29.79 2 5.581 P 0.018 : A final staining score 4 was defined as overexpression, and a final staining score < 4 was defined as nonoverexpression. doi: 10.1371/journal.pone.0084735.t001 snail increased, whereas the expression of E-cadherin decreased in the Beas2B cells transfected with the pcDNA3HA-RBP2 21363929 plasmid. Meanwhile, both the A549 and SK-MES-1 cells exhibited lower levels of RBP2, N-cadherin and snail proteins and higher level of E-cadherin protein when transfected with RBP2 siRNA2. Similarly, real-time PCR analysis further showed that the RBP2, N-cadherin and snail mRNAs increased and the level of E-cadherin mRNA decreased in the Beas2B cells treated with the pcDNA3-HARBP2 plasmid. Additionally, lower levels of RBP2, N-cadherin and snail mRNAs and a higher level of E-cadherin mRNA were observed in both the A549 and SK-MES-1 cells that were treated with RBP2 siRNA2. Effects of RBP2 on the E-cadherin promoter Because Huang et al. confirmed that RBP2 could directly bind to the promoter region of E-cadherin using a CHIP assay, we examined the activity of the same Ecadherin promoter region using a luciferase assay in the A549 and Beas2B cells. Therefore, we hypothesized that RBP2 regulated N-cadherin and snail through the activation of Akt signaling, and we examined the levels of p-Akt and Akt in the A549 cells using western blot analysis. The results showed that p-Akt protein was high in the A549 cells and decreased after the depletion of RBP2. The expression of p-Akt was positively correlated with the expression of RBP2. Next, to confirm the effects of Akt signaling on the expression of N-cadherin and snail, we treated A549 1659286 cells with a PI3K inhibitor for 24 h and examined the levels of the N-cadherin and snail proteins. LY294002 has been shown to block PI3K-dependent Akt phosphorylation and kinase activity. Interestingly, the levels of both N-cadherin and snail declined. Therefore, the expression of N-cadherin and snail was positively associated with the expression of p-Akt. Discussion RBP2, a newly identified histone demethylase, belongs to the JARID family and possesses an ARID. It often occupies and regulates the promoters of multiple genes that contain the H3K4me3. More importantly, RBP2 is believed to participate in many cell biological functions, especially in tumor biology. Our study reveals a novel insight into the AEB 071 pathophysiology of EMT, and we provide evidence that RBP2 induces EMT in NSCLC. RBP2 plays an important role in human cancer. For instance, the overexpression of RBP2 inhibits the senescence of gastric cancer cells. The depletion of RBP2 impairs proliferation but promotes senescence and differentiation in mice lacking Men1 and Rb1. Drug tolerance of lung cancer cells requires RBP2. Knockdown of RBP2 led to increased levels of H3K4me3 at the promoters of the DAF and HMOX1 genes in the Beas2B cells. RBP2 up-regulates the expression of cyclin D1, cyclin E1 and integrin 1 to enhance cell proliferation, migration and invasion. In this paper, we also detected the expression of RBP2 in NSCLC tissues and analyzed the relationships between RBP2 and each of the clinicopathological features of NSCLC. The results showed that RBP2 was overexpressed in human NSCLC but there was no significant relationship between the overexpression of RBP2 and each clinicopathological feature. Additionally, the effects of RBP2 on the migration of lung cancer cells were studied, and we found that RBP2 could enh