Figure 2From: OncomiR-196 promotes an invasive phenotype in oral cancer through the NME4-JNK-TIMP1-MMP signaling pathwayNME4 is a direct regulatory target of miR-196. (A) Differential expression of miR-196a/b and NME4 mRNA in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-qPCR. Relative levels of miR-196a/b were determined after normalization to the U6 RNA level (internal control) for each sample. The NME4 RNA level was normalized to actin for each sample. (B) Differential expression of NME4 mRNA and protein in two immortalized normal keratinocyte and four oral cancer cell lines, as determined by RT-PCR and western blotting. Actin expression was also determined as an internal control. (C) Inverse correlation between NME4 and miR-196a/b expression which determined by RT-qPCR. (D) Effect of miR-196 modulation on NME4 mRNA expression. OECM1 and SAS cells transfected with miR-196a/b–specific antagomirs or overexpression plasmids were harvested. Total protein was extracted and subjected to western blot analysis of NME4 expression. The relative expression was determined after normalization to actin as an internal control. (E) The mature sequences of miR-196a and miR-196b, as well as the 3′-UTR sequence of the human NME4 gene, are shown. The mutant sequence of the 3′-UTR region of NEM4 was designed. Luciferase reporter assay was performed to determine whether NME4 is a direct miR-196a/b target gene. Cells transfected with miR-196–specific antagomirs or overexpression plasmids were co-transfected with pMIR, p-UTR-WT, or p-UTR-mut. Renilla luciferase was also transfected as a reference control for each condition. Firefly and Renilla luciferase activities were measured using the dual-luciferase reporter assay. (*p < 0.05 , **p < 0.01, ***p < 0.005, t-test).Back to article page