R 196b at 12 hours. Consist ently, miR 196 over e pression significantly enhanced cell migration in both cell lines, with 1. CC-5013 9 2. 7 and 1. 7 2. 2 fold increases, respectively, for miR 196a and miR 196b at 9 hours. Similar effects were also observed in cell invasion ability. Depletion of miR 196a or miR 196b dramatically reduced the invading cells by 40 50% in both OECM1 and SAS cells. Consistently, miR 196a or miR 196b over e pression significantly enhanced cell invasion by 2. 2 fold in both cell lines. Supporting these cellular findings, the e pressions of cell adhesion molecules N cadherin and fibronectin were up regulated in the miR 196 over e pressing cells. Collectively, miR 196a and miR 196b promote migration and invasion in oral cancer cells but e hibit minimal effects on cell growth.

NME4 is a direct regulatory target of miR 196 To identify the potential target of miR 196, computa tional prediction software, including PicTar, miRanda, and TargetScan, was used. These programs identified common candidates for both miR 196a and miR 196b. The e pression of these genes was further confirmed by RT qPCR in response to miR 196 modulation. Four genes were upregulated by more than 20% after miR 196 depletion, whereas three genes were downregulated by more than 20% after miR 196 overe pression. Of these genes, only the e pression of NME4 changed consistently in both confirmation studies. Therefore, NEM4 is a potential miR 196 regulatory target. To determine the association of miR 196 and NME4, the e pression levels of these molecules were e amined in two lines of normal keratinocytes and four oral cancer cell lines.

As shown in Figure 2A, miR 196 was signifi cantly up regulated in all cancer cell lines compared to those in normal cells, with 92 and 71 fold higher in average for miR 196a Dacomitinib and miR 196b respectively. By contrast, NME4 e pression was reduced in all cancer cell lines at both mRNA and protein levels. This reverse correlation between these molecules further suggests that NME4 is a regulatory target of miR 196. To further e amine whether NME4 is a down stream target of miR 196, the potential effect of NME4 protein e pression was determined in response to miR 196 modulation. As shown in the Figure 2D, NME4 levels were elevated or reduced upon miR 196 silencing or over e pression. To validate the regulatory target of NME4, a luciferase reporter assay was performed.

Reporter plasmids that carry human NME4 3UTR wild type sequence and mutant sequence were co transfected with either miR 196 antagomirs or e pression plasmids. Silencing miR 196a www.selleckchem.com/products/FTY720.html or miR 196b in creased NME4 wild type UTR reporter activity in both OECM1 and SAS cells but had no effect on mutant UTR or empty vector reporter activity. Consistently, over e pression of miR 196a or miR 196b reduced NME4 wild type UTR reporter activity both cell lines. However, these miR 196 modula tions e hibited minimal effects on mutant UTR reporter activity. Taken together, these results sug gest that N